A fish market survey using a novel PCR-sequencing-based protocols for the identification of commercial significant fish species

This study developed a simple, specific, and affordable PCR-sequencing- COI gene-based protocol for the simultaneous identification of some important commercial fish species: Merluccius merluccius , Lates niloticus , Gadus morhua , Ruvettus pretiosus , Pangasianodon hypophthalmus , Epinephelus spp. For this study, a local market survey on fish was carried out to evaluate the application of labelling laws and to detect fraudulent actions using the developed PCR protocols. Ten specimens of each fish species of interest were obtained from wholesale fishery plants and were utilized for the protocol development. DNA was extracted from the individual samples and quantified. DNA isolates were subjected to end-point PCR and the PCR products were sequenced. For the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software “Primer-BLAST” to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within the COI gene for M. merluccius , L. niloticus , G. morhua , R. pretiosus , P. hypophthalmus , Epinephelus spp., respectively. Single PCR was performed using DNA isolates and developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected sequences of the COI gene and showed a similarity ranging from 99 to 100%. Among 43 samples obtained for the survey, 19 (44.2%) were mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and fish marketplaces and 1 (2.32%) from hypermarket stores. Among fish samples purchased at local fisheries and fish marketplaces, fraudulent actions were observed more frequently in fish slices (100%) than fish fillets (65%). Regarding fish fillets, out of four samples labelled as grouper, three were L. niloticus and one P. hypophthalmus . Two fillets marketed as cod were substituted with L. niloticus . Five samples labelled as “fillet” and two samples labelled as “perch” were identified as P. hypophthalmus . Regarding fish slices, all samples marketed as grouper ( E. marginatus ) were slices of R. pretiosus . The single case of mislabelling detected from fishery products purchased at hypermarket stores was a sample of “Spinycheek grouper” ( Epinephelus diacanthus ) that was indicated on label as “Grouper” ( Epinephelus marginatus ). In summary, our work highlights the need for continuous surveillance of the commercialization of fishery products, to reduce the number of fraud cases that happen in the market. Furthermore, our protocols based on PCR techniques could be useful for quality control of fresh finfish and to strengthen controls on the most frequent fraudulent actions of marketed fishery products.


INTRODUCTION
The seafood consumption has increased several folds during the last 50 years, including wild and aquaculture products [1]. The species substitution in which low value fish is replaced with high value ones is a prominent phenomenon in the international seafood trade and is a leading cause of fraud in the fishery sector, leading to economic and health concerns. Fishery products present a valid alternative to other types of animal-origin food (terrestrial animal meat, eggs, dairy products, etc.) especially for their high digestibility due to the lower presence of connective tissues and lipid components [2], [3], [4]. Despite its increasing popularity, seafood is one of the prominent products associated with food frauds. Authentication studies and market monitoring of commercial Design of the experiment: To develop novel protocols based on the PCR for the genetic identification of some significant commercial fish species, we created specific primers for the identification of the following: Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasianodon hypophthalmus and Epinephelus spp. Mitochondrial cytochrome c oxidase subunit 1 (COI) gene was selected to identify fish species. This genetic fragment presents very low intraspecific variability, thus permitting the unequivocal identification of fish species. Then, we applied the developed protocols to a local survey and ascertained fishery products' correct labelling at local retail outlets.
Primer design: Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasianodon hypophthalmus and Epinephelus spp. were the six fish species of interest subjected to the study. Mitochondrial cytochrome oxidase subunit 1 (COI) gene was used to identify the above fish species. For each fish species, COI sequences were obtained from the GenBank database and aligned and compared by the program BioEdit. The primers were developed via two methods. Firstly, species-specific primers to amplify fragments of 200 bp, 250 bp, 300 bp, 350 bp and 400 bp within the COI gene for M. merluccius, L. niloticus, G. morhua, R. pretiosus, P. hypophthalmus, respectively, were designed by the program "Primer Express 3.0". The program "Primer Express 3.0" was set according to the parameters reported in Table 1. Secondly, COI, FASTA sequences for Epinephelus spp. and G. morhua were inserted in the software "Primer -BLAST" to develop primers to amplify fragments of 522 bp and 562 bp, respectively. The software "Primer -BLAST" was set to create primers according to the parameters reported in Table 2.  . Samples purchased at fisheries and fish marketplaces consisted of 20 fish fillets and 5 fish slices. Regarding fish fillets, four were labelled as grouper, two as cod, three as Nile perch, four as striped catfish, five reported as "fillet" from local fisheries and fish marketplaces and two as "perch" (both without the indication of fish species). All fish slices were labelled as grouper (Table 7). After collection samples were subjected to DNA extraction with NucleoSpin Tissue Kit (Macherey-Nagel). All DNA samples were quantified (about 20 ng/μL) by Nanodrop (Thermo Scientific) and subjected to PCR with original species-specific primers developed for the identification of fish species. Primers were commercially synthesized by Sigma Aldrich (Milan, Italy). Primers were diluted to a final concentration of 100 nM. PCR primers for each fish species of interest were created. Both methods developed two pairs of primers for G. morhua (Table 3).     (Table 5). The PCR amplicons were analysed by agarose gel electrophoresis by using a horizontal 2% (wt/vol) agarose gel in lX TBE buffer (pH 8.3; 0.09 M Tris, 0.09 M boric acid, 2.0 mM EDTA) and with 0.003% (wt/vol) ethidium bromide for DNA staining. PCR products were mixed with a sample buffer of 1X TBE and then applied to each well. Gel ran in 1X TBE buffer at 200 V for 30 min. The DNA marker used was Amplisize molecular ruler, 50% GC content, 50-2000 bp, 10 bands (Bio Rad, Hercules, Spain). The PCR products were visualized and photographed by a Gel Doc XR+ System transilluminator (Bio Rad, Milan, Italy). Sequencing: PCR products were purified using Montage PCR filter units (Millipore, Milan, Italy) and sequenced by BigDye 3.1 Ready reaction mix (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions (Tables 4 and 5). Sequences were imported and assembled with the BioNumerics 7.5 software (Applied Maths, Saint-Martens-Latem, Belgium) and searched for homologous sequences by BLAST search analysis (http://www.ncbi.nlm.nih.gov). Multiplex-PCRs: Primers were developed to obtain amplicons with different lengths (at least 50 base pairs). Duplex and Triplex PCR protocols were developed to simultaneously analyse more fish species using the designed primers with several combinations (Table 6).

Statistical Analysis
Statistical Analysis is not required for this study.

RESULTS AND DISCUSSION
Specificity of the developed protocols PCR assay allowed the detection of DNA extracted from all specimens of each fish species of interest, giving fragments of the expected length. At the end of the running, the electrophoresis agarose gel showed a clear separation of amplicons due to their different sizes (Figures 1, 2, 3, 4 and 5). Single PCRs performed for the specificity tests gave the expected results. After sequencing, the isolates were compared with the selected sequences of COI gene and showed a similarity ranging from 99 to 100%. Grouper samples subjected to Epinephelus spp. authentication, showed 97.5% homology to Epinephelus costae GenBank entry (KM077928.1) and 100% homology to Epinephelus marginatus GenBank entry (KC500692.1). The results of the experiment are shown in Table 1 and Figure 1.

Identification of the samples used for the survey
Overall, out of 43 fish samples analysed, 19 (44.2%) resulted mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and marketplaces and 1 (2.32%) from hypermarket stores ( Table 7) showing an amplicon of 522 bp. To ascertain the existence of false positives, identifications were confirmed by sequencing. After sequencing, Epinephelus spp. isolates showed 100% homology to Epinephelus diacanthus GenBank entry (EF609520.1). Out of 25 fish samples purchased at fisheries and fish marketplaces, 18 (72%) were mislabelled. Cases of mislabelling regarded more fish slices (100%) than fillets (65%). Regarding fish fillets, three Nile perch fillets (15%) and four striped catfish fillets (20%) were correctly labelled. The DNA analysis on the remaining fillets showed that thirteen samples were mislabelled (65%). All samples marketed as grouper fillets showed fraudulent actions. In fact, out of four samples labelled as grouper, three (75%) tested positive for L. niloticus showing an amplicon of 250 bp., and one (25%) positive for P. hypophthalmus showing an amplicon of 400 bp. Both cod fillets (100%) resulted to be L. niloticus, showing an amplicon of 250 bp. The 5 samples from local fisheries and fish marketplaces labelled as "fillet" and the 2 samples labelled as "perch" were identified as P. hypophthalmus showing an amplicon of 400 bp (Table7). As regards grouper slices, all samples (100%) showed fraudulent species substitutions; in fact, R. pretiosus was marketed as grouper (amplicon of 350 bp.). Table 7 Results of the survey on the application of the labelling laws and for the detection of fraudulent actions.  [26]. In the fish sector, the identification of fish species throughout the production chain is of main importance, even if fishery products have already been processed. In fact, there are different ways to purchase fish and fishery products: whole, fillets, slices, skewers or mixed with other species for gastronomic dishes (seafood salad, risotto mix, fish fingers, etc.). Furthermore, the presence of similar fish species, but very different from a nutritional and organoleptic perspective, is more frequent. Currently, commercial fishery products in Europe come from all parts of the world, meaning that accurate species identification is not always easy. In this situation, both sanitary and quality control and product traceability could be obstructed because fish are not easily identifiable, with the increase in commercial (aliud pro alio) and sanitary frauds (commercialisation of toxic organisms). Further, food poisoning due to the consumption of toxic fishery products belonging to Tetradontidae, Molidae, Diodontidae and Canthigasteridae families may occur [27], although their marketing is forbidden by European Regulations (EC Reg. 853/2004). For example, oilfish (Ruvettus pretiosus) is seldom marketed in conformity with the current EU Regulation (EC Reg. 1021/08) and it is often commercialized in place of the most popular, expensive, and precious species, such as grouper (Epinephelus spp.). The problem of fraudulent actions in the commercialization of foods is strongly felt at the European Union level; in fact, recently a recommendation was enacted in the need to establish a "coordinated plan of supervision designed to determine the prevalence of fraudulent practices in the marketing of certain foodstuffs", including fishery products (EU Recommendation n. 1558 -12 March 2015). EU enforced the Regulation (EU) 1379/2013 that lay down to the fish economic operators to report on the label of the fish products some information such as the commercial and scientific designation name, the production method (catch or breeding), its origin (the FAO fishing area for sea products and the name of the Country for breeding products) and the fishing gear [28]. The objective of this regularity policy was to generate safe supply for consumers and the food processing industry as well as to give consumers a more detailed information about food products to protect fraud, prevent illegal fishing and promote sustainable aquaculture. Similar regulations are in force in many countries; however, despite these regulations, widespread seafood mislabelling has been identified in the United States and Canada [16], [17], In Europe [14], Asia [9] and south Africa [19] indicating the need for stringent control measures to generate efficient species identification [20]. In fact, in 2016, Oceana published a major report by reviewing more than 200 published studies across 55 countries and found 20% mislabelling in catering and related sectors [20]. In 2021, Oceana Canada observed 46% mislabelling in seafood products, which is just 1% less compared to a study conducted during 2017-2019 [29]. In 2021, a Guardian Seascape analysis of 44 recent surveys of more than 9,000 seafood samples from restaurants, fishmongers, and supermarkets in more than 30 countries conducted and found that 36% samples were mislabelled, exposing a large amount of seafood fraud at global scale [30]. All [11] show the concerns related to fish fraud [33], [34], [35]. The development of PCR protocols has allowed a rapid and specific response for identifying fish species. In fact, the time required from the arrival of the fish sample to the end of the analysis was about 6-8 h. Thanks to the development of Duplex and Triplex PCR protocols, additional information may be gained from a single test run with considerable saving of time, reagents, and efforts within the laboratory. Furthermore, the applicability of the assay to commercial fishery products has been demonstrated. In fact, in our survey, of the 43 investigated samples, we detected 19 (44.2%) mislabelled samples. Most of the mislabelled samples derived from local fisheries and marketplaces (41.9%) and one sample (2.32%) from hypermarket stores. Our findings are similar to the results obtained from a national seafood fraud investigation carried on in the United States from 2010-2012. In this survey, out of 1200 seafood samples from 674 retail outlets in 21 States, DNA testing found that one-third (33 per cent) were mislabelled [36]. Forty-four per cent of the retail outlets visited sold mislabelled fish. Also, a recent Italian investigation revealed numerous commercial frauds; for example, Cutarelli et al., found that a sample marketed as "frozen grouper fillet" was made from halibut (Hippoglossus hippoglossus) instead of grouper (E. marginatus) [37]. Given consumers' high demand for grouper, the prices at the subsequent wholesale and retail market levels are also high relative to other finfish species. Additionally, the importation of large quantities of grouper from many foreign sources must meet the ever-growing demand for grouper. The strong demand for grouper and its high market value, which continues to be evident in the market, is also a motivation for economic frauds. The most prevalent economic fraud associated with grouper is the selling of a cheaper finfish as grouper. In fact, the most common types of mislabelling among the grouper samples collected in the US were substitutions with farmed Asian striped catfish (Pangasianodon hypophthalmus), freshwater perch (Macquaria novemaculeata), weakfish (Cynoscion regalis), bream (Abramis brama), and king mackerel (Scomberomorus cavalla). It is important to underline that grouper is a precious fish species often an item of fraud; in fact, when grouper is sold as fillet, its main features completely disappear, and its identity cannot be established on the basis of morphological features [38]. A survey carried out by the Eurofishmarket (www.ilfattoalimentare.it) showed that around 15% of fresh/frozen grouper fillets sold on the market belonged to other species. These facts are strongly confirmed in our survey, in fact, we found that all samples marketed as grouper slices (E. marginatus) were slices of R. pretiosus. Such fraud could be considered both a commercial and a sanitary fraud because R. pretiosus is a fish known for its potential dangerousness for consumer. In fact, R. pretiosus, also known as "oilfish," is a deep-sea fish that stores a large amount of wax esters in its body for buoyancy control. In humans the accumulation of the indigestible wax esters in the rectum through the consumption of these fish produces discharges or leakage per rectum as orange or brownish green oil, but without noticeable loss of water; this response is called keriorrhea [39]. Outbreaks of keriorrhea have been repeatedly reported across continents. In the EU, the marketing of R. pretiosus is regulated by the EC Reg. 1021/08 (EC Reg. 1021/08). According to this regulation, food business operators must sell oilfish products in packaged form and provide information on label to the consumer about their gastrointestinal adverse effects.

Retail outlet
In conclusion, our method based on PCR constitutes an effective molecular tool for detecting fraudulent substitution of fish species of interest applicable to raw finfish. These protocols could be applied to both quality control and official sanitary control of fishery products and to help the anti-fraud actions control fishery products' traceability and labelling.

CONCLUSION
The seafood industry is one of the imported traded products globally. Due to several health benefits, availability, less religious concerns, and possibility options, its demand and consumption increased exponentially. Due to increasing trade value, it is continuously vulnerable to frauds where costly fish can be replaced with cheap fishes, particularly in products where morphological identification is lost. Fish frauds may have health and environmental concerns. It must be authenticated before serving customers. Due to the limitations of protein-based methods, DNA-based methods like multiplex PCR provide a better alternative for species identification and tracking food fraud. It can help food control authorities to ensure food safety and the rights of consumers.