Introduction
The red porgy is a benthopelagic species of high commercial value, widely traded in the Greek market. Its wild populations are located the Mediterranean Sea, but also in the Atlantic coasts of America, South Europe and Africa (Fishbase). Wild Pagrus pagrus from fisheries in Greece, shows similar morphological features with the corresponding imported, and closely-related species of Sparidae, as Pagellus erythrinus, and Dentex gibbosus. Pagrus sp. is reared in Greek fish farms, with its production conquering the third place among reared species in Greece. Pagrus fish farming products sold in the Greek market are found under various names, with the majority reported as Pagrus major. The trading of wild, reared, or imported representatives in the market, raises the question of authenticity and proper labeling of Pagrus products. Although fish can be classified by their morphological characteristics, molecular techniques based on genetic material (DNA), are currently being used for accurate and robust species identification. Having the ability to cross-check the sequences with databanks, the genetic authenticity and origin of each sample can be assessed, thus contributing to the detection of mislabeling and fraud. The mitochondrial DNA (mtDNA) is a useful tool for investigating molecular mechanisms and evolutionary relations between different fish species (Ceruso et al. 2019). The gene region used as DNA barcode in species identification (Ratnasingham 2007), is part of the cytochromic oxidase subunit I gene (COI). However, for the discrimination between closely related species or populations within species, data could also be drawn from other mtDNA regions (cytb, 16sRNA, D-loop etc.). Up to date, the complete mitochondrial genome of P. major, P. auriga and P. caeruleostictus have been determined. In this study, the complete mtDNA of Pagrus pagrus, captured in the central Aegean Sea, was sequenced. Data inferred from this sequence along with barcoding of samples collected in the Greek market (locally fished, reared, imported) were used in different molecular techniques, in order to establish a fast and reliable method for species identification within Pagrus genus and for estimation of population differentiation within wild Pagrus pagrus.
Materials and Methods
The specimens included wild P. pagrus (70 samples) caught in the Greek seas, FAO subareas 37.2 (Ionian Sea division 37.2.2) and 37.3 (Aegean and Cretan Seas), farmed Pagrus (15 samples from various Greek fish farms) and imported (labelled as P. pagrus), purchased from fish markets and supermarkets (10 samples). Fish species were identified according to their morphological characteristics and categorized in groups by their origin. Total DNA was extracted from the skeletal muscle or liver using NucleoSpin® Tissue Macherey-Nagel, according to the manufacturer’s instructions. For the determination of total mtDNA sequence, a specimen caught at Central Aegean FAO division 37.3.1 was selected. Primers were either designed or selected from the literature (Miya and Nishida 2000), for the amplification of total mtDNA in 42 overlapping fragments ranging from 300-4.500 bp. Both mtDNA strands were completely sequenced. For barcoding, a fragment of 648 bp of COI was amplified using universal primers (Ward et al., 2005). In order to distinguish P. pagrus populations, Forensically Informative Nucleotide Sequencing (FINS) was carried out for cytochrome b gene. A 583 bp fragment was amplified using primers designed in this study. Sequences were identified with Blast search tool, compared and aligned with sequences from BOLD database and aligned with clustalW2 (Sievers et al. 2011). After in silico analysis, 3 fragments were chosen for Restriction Fragment Length Polymorphism analysis (PCR-RFLP) using a set of 10 restriction enzymes. Smaller fragments of COI and cytb (100-300 bp) were used in Real-time PCR (qPCR) and High-Resolution Melting (HRM) analysis.
Results
The complete mtDNA of Pagrus pagrus has a size of approximately 17 kb and revealed the same structure as all mtDNAs of teleosts, containing 13 protein, 22 tRNA and 2 rRNA genes and two non-coding regions (D-loop and L-origin of replication). Gene arrangement was typical of Sparidae species, with most of the genes encoded by the heavy strand, except for the NADH dehydrogenase subunit 6 (ND6) and eight tRNAs that are encoded by the light strand. The results showed that there is a clear distinction between wild specimens, imported and reared ones. According to homology in FishBol, all specimens from fisheries or imported were identified as P. pagrus and all specimens from Greek fish farms as P. major. Sequences of the same samples that were analyzed for cytb (FINS), confirmed these findings, being more indicative for differences between populations. Results from RFLP showed a pattern that could distinguish between P. pagrus and P. major but not between Greek and imported P. pagrus. Preliminary results of qPCR and HRM analyses from regions of COI, and cytb, are indicative and promising for fish species and population identification.
Conclusions
Fisheries plays an important socioeconomic role in Greece, providing the market with products of high nutritional value. Due the high price of red porgies, it is important to conserve and validate the value of these products. The detection of P. major as a common species in Greek porgy aquaculture today raises the questions about the environmental impact from the rearing of an alien species (escapes, mixes with wild populations) in the Mediterranean and authenticity issues as described in Regulation (EU) No 1379/2013. In order to enable consumers to make informed choices, it is necessary to provide clear and comprehensive information on, inter alia, the origin and the method of production of the products. P. pagrus, fished in Greece, show similar morphological features with the corresponding imported, farmed fish of the same genus and closely-related species of Sparidae. Furthermore, in case the fish has been processed (exfoliated, fillet or cooked), even classification experts are unable to identify the sample. Data of this study, will ensure the identity, originality and authenticity of Greek fishery products. Moreover, extended sequence data are already being used for the development of a methodology (qPCR, HRM) that will provide a rapid tool against fish mislabeling and phenomena of fish fraud.
Acknowledgements
This work is supported by the Greek Operational Programme Maritime & Fisheries 2014-2020 (MIS 5033599), which is co-financed by the EU- European Maritime and Fisheries Fund and the Hellenic Republic Ministry of Rural Development and Food.
References
Ceruso M., Mascolo C., Anastasio A., Pepe T. & Sordino P. 2019. Frauds and fish species authentication: Study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers. Food Control 103, 36–47.
Miya M. & Nishida M. 2000. Use of Mitogenomic Information in Teleostean molecular Phylogenetics: A Tree-Based Exploration under the Maximum-Parsimony Optimality Criterion. Molecular Phylogenetics and Evolution 17, 437-455
Ratnasingham, S. & Hebert P.D.N. 2007. BOLD: The Barcode of Life Data System (www.barcodinglife.org). Molecular Ecology Notes 7, 355-364.
Sievers F., et al. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7, 539.
Ward R.D., Zemlak T.S., Innes B.H., Last P.R. & Hebert P.D.N. 2005. DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360, 1847–1857.