Molecular identification of Atlantic goliath grouper Epinephelus itajara (Lichtenstein, 1822) (Perciformes: Epinephelidae) and related commercial species applying multiplex PCR

The Atlantic goliath grouper, Epinephelus itajara, is a critically endangered species, threatened by illegal fishing and the destruction of its habitats. A number of other closely related grouper species found in the western Atlantic are also fished intensively. While some countries apply rigorous legislation, illegal harvesting followed by the falsification of fish products, which impedes the correct identification of the species, is a common practice, allowing the catch to be marketed as a different grouper species. In this case, molecular techniques represent an important tool for the monitoring and regulation of fishery practices, and are essential for the forensic identification of a number of different species. In the present study, species-specific primers were developed for the Cytochrome Oxidase subunit I gene, which were applied in a multiplex PCR for the simultaneous identification of nine different species of Epinephelidae: Epinephelus itajara, E. quinquefasciatus, E. morio, Hyporthodus flavolimbatus, H. niveatus, Mycteroperca acutirostris, M. bonaci, M. marginata, and M. microlepis. Multiplex PCR is a rapid, reliable and cost-effective procedure for the identification of commercially-valuable endangered fish species, and may represent a valuable tool for the regulation and sustainable management of fishery resources.


Introduction
The increasing demand for fishery resources has led to the gradual, worldwide collapse of stocks in marine ecosystems, leading to the substitution of preferred species by smaller, short lived ones, in increasingly simplified trophic networks (Pauly et al. 2002).These larger-bodied, preferred species include groupers (Epinephelidae) and snappers (Lutjanidae), which are associated with reef habitats, are highly valued commercially, but are extremely vulnerable to fishery pressure (Begossi et al., 2012a).
Epinephelidae is a group of reef fishes comprising more than 160 species distributed in 16 genera.The greatest diversity of epinephelids is found in the Indo Pacific region, and the eastern Atlantic is the probable center of dispersal of all the epinephelid groupers in the mid-Eocene (Ma, 2014).The present day fauna of the western Atlantic includes about 25 species of the family Epinephelidae, arranged in eight genera (Craig et al., 2011).
The Atlantic goliath grouper, Epinephelus itajara (Lichtenstein, 1822), is the largest grouper species (> 400 kg) found in the Atlantic Ocean, and is listed by the IUCN as "critically endangered", due to population decline resulting from overfishing and habitat degradation (Craig, 2011;IUCN, 2015).This grouper is found in tropical and subtropical waters of the western Atlantic, between Florida and southern Brazil, including the Gulf of Mexico and the Caribbean, as well as the west coast of Africa, between the Congo and Senegal (Francesconi & Schwartz, 2000;Craig et al., 2011).
Despite the prohibition of fishing in 2002, the capture and sale of Atlantic goliath grouper has been recorded frequently by the authorities, with a mean annual catch of more than 330 tons between 2003 and 2011 (IBAMA, 2004;2005;2007a;2007b;2008;MPA, 2012a;2012b;2012c).Considering also the lack of data on artisanal fisheries, together with the common practice of disguising catches, it seems likely that the Brazilian populations of Atlantic goliath grouper are impacted severely by fisheries, irrespective of the established conservation legislation.
Genetic tools for the molecular identification of a number of different fish species have been developed during the past decade (Ward et al., 2005;Craig et al., 2009;Hashimoto et al., 2010;Rodrigues et al., 2011;Alexandre de-Franco et al., 2012;Ulrich et al., 2013;Veneza et al., 2014).Carvalho et al. (2015) has shown the DNA barcoding to be an effective molecular tool for the detection of commercially-valuable species such as the groupers, while Torres et al. (2013) successfully applied PCR RFLP and SNP detection techniques to the identification of specimens of E. itajara, E. morio, M. bonaci, and M. marginata.The multiplex PCR technique, based on the COI gene (De-Franco et al., 2010;Mendonça et al., 2010) and other mitochondrial (Trotta et al., 2005;Hashimoto et al., 2010;Ravago-Gotanco et al., 2010) and nuclear genes (Shivji et al., 2002;Chapman et al., 2003;Rodrigues et al., 2011), has proven to be an efficient and rapid low-cost tool that is safe and easy to apply (Abercrombie et al., 2005).This technique permits the identification of illegal fishing by the authorities, even when the catches have been processed for consumption, and contribute to conservation practices on a worldwide scale (Mendonça et al., 2010).
Large-bodied fishes such as groupers (Epinephelidae), which inhabit reef ecosystems around the world, have a high commercial value and are intensively exploited, although their biological characteristics, such as their longevity, late sexual maturation, and breeding aggregations, that make them especially vulnerable to overfishing (Sadovy & Eklund, 1999;Chiappone et al., 2000;Pauly et al., 2002;Sadovy de Mitcheson et al., 2013).Industrialization of fisheries has led to a sharp decline in the marine wildlife, which will require the adequate management and controls to ensure that currently environmental alterations do not result in a complete collapse of fishery stocks (McCauley et al., 2015).
Considering the urgent need to conserve groupers stocks and the importance of identifying these species where illegal harvesting is disguised by the falsification of fish products, the present study aimed to develop a rapid, simple, and low-cost procedure characteristics of the COI gene to discriminate nine species of epinephelids.
The specimens obtained from fish markets at a number of different localities around the coast of Brazil, and were captured by fishing vessels operating off the coast of Santa Catarina, Espírito Santo, southern Bahia, and Paraíba.The specimens of E. itajara were obtained from the states of Pernambuco, Sergipe, Bahia, Espírito Santo, and Santa Catarina using traditional fish traps, with samples of the tail fin being extracted using a non-lethal procedure.Once the samples were collected, the E. itajara specimens were weighed, measured, marked, and released back into the area where they were caught.The samples were preserved in 95% ethanol and stored in a freezer at -20 o C.
Given the evolutionary proximity of the Atlantic and Pacific goliath groupers (Craig et al., 2009), samples of Epinephelus quinquefasciatus (Bocourt, 1868), which is endemic to the eastern Pacific, were obtained from fisheries in Puerto Sandino, Nicaragua.These samples were used to validate the results of the multiplex PCR and guarantee the reliability of the analysis of the epinephelid species.
Extraction, Amplification and Sequencing of the DNA.The genomic DNA was extracted using the method described by Aljanabi & Martinez (1997).The amplification reactions for the COI were run in an Applied Biosystems® Veriti® 96 Well Fast thermal PCR cycler, using 25 μl of solution containing 0.8 mM of dNTP (Invitrogen®), 1.5 mM of MgCl 2 , Taq DNA polymerase enzymatic buffer (Tris-HCl 20 mM at pH 8.4 and KCl 50 mM), 1 unit of Taq Polymerase enzyme (PHT Phoneutria®), and 5µM of each universal primer, F1 5" TCA ACC AAC CAC AAA GAC ATT GGC AC 3" and R1 5" TAG ACT TCT GGG TGG CCA AAGAAT CA 3" (Ward et al., 2005).Each PCR amplification cycle consisted of denaturation at 96ºC for 30 s, hybridization at 52ºC for 30 s, and extension at 68ºC for 1 min, repeated 35 times.The DNA segments were visualized in agarose gel in TAE 1X (Tris-Acetate EDTA) using GelRed (Biotium®) under a MacroVue UV-25 (Hoefer®) ultraviolet transilluminator.Sequencing was conducted using the BigDye Terminator kit, according to the maker's recommendations (Applied Biosystems®).The samples were precipitated with 0.7 uL of EDTA (125mM), 0.7 uL of sodium acetate (3M) and 25 uL of ethanol (100%), then washed in 35 uL of 70% ethanol and the containers were dried at 95 o C for 8 minutes before sequencing in the ABI Prism 3130 (Applied Biosystems®) Multiplex PCR.Nine species-specific primers were designed based on the exclusive polymorphic sites of the COI gene found in each of the study species (Table 1), which were used in combination with the primers F1 and R1 (Ward et al., 2005).All the COI amplification reactions based on the species-specific primers were run in a thermal cycler with 25 μl of solution containing 0.8 mM of dNTP (Invitrogen®), 1.5 mM of MgCl 2 , Taq DNA polymerase enzymatic buffer (Tris-HCl 20 mM at pH 8.4 and KCl 50 mM), 1 unit of Taq Polymerase enzyme (PHT Phoneutria®), and 5µM of each specific primer.The amplification and visualization of the sequences followed the same protocol used for the F1 and R1 primers.
Analyses.The sequences were aligned using the Muscle program, available online at www.ebi.ac.uk/Tools/msa/ muscle/ (Edgar, 2004).The taxon´s phylogram was analyzed in Mega 5.2 (Tamura et al., 2011) using the Neighbour Joining (NJ) method based on the Kimura 2 parameter model (Kimura, 1980), and tested using the bootstrap method with 1000 pseudoreplications (Felsenstein, 1981).The identification of the species based on the COI sequences was confirmed by blasting against the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).To guarantee the quality of the haplotypes, they were blasted using MegaBLAST of the GenBank database to minimize the use of sequences with inverted amplification of translocated nuclear copies of the mitochondrial DNA (numts), as recommended by Song et al. (2008), with only the sequences that were 99% or 100% similar to the species analyzed.
Table 1.Species-specific primers developed for the present study, the size of the fragments isolated, and the GenBank accession numbers of the sequences obtained for the nine grouper species of the family Epinephelidae. 1. Epinephelus quinquefasciatus 2. Epinephelus morio 3. Epinephelus itajara 4. Hyporthodus flavolimbatus 5. Mycteroperca microlepis 6. Mycteroperca bonaci 7. Mycteroperca marginata 8. Hyporthodus niveatus 9. Mycteroperca acutirostris.Phylogram included sequences from the GenBank database of other species of commercially-important reef-dwelling fishes belonging to the families Epinephelidae [e.g., Cephalopholis fulva (Linnaeus, 1758) and Hyporthodus nigritus (Holbrook, 1855)], and Polyprionidae [e.g., Polyprion americanus).These species were included to certify the separation of the specific taxa identified from the samples collected during the present study.Polyprion americanus is an endangered species, which was included in the tree because it has been identified in categories of fish similar to the other epinephelid species (Asensio, 2008;Bertoncini et al., 2013).

Species
The set of primers designed during this study amplified fragments of different sizes (number of base pairs) of the mitochondrial DNA for each of the epinephelid species.In the multiplex PCR, the species specific primers (forward) designed in the present study were used together in the same reaction with the universal primers (F1 and R1) described by Ward et al. (2005).The size of the fragments produced for each species is shown in Table 1.
The species-specific primers were initially tested on a set of species samples to test their effectiveness for a standard PCR.The sequences amplified corresponded to the primers designed for each species.The different groups of samples were then tested together to identify possible incorrect amplifications.

Results
Amplification of the COI gene resulted in a fragment of 655 base pairs (bp) in all nine epinephelid species analyzed, with a mean nucleotide composition of 31.0%Thymine (T), 27.1% Cytosine (C), 24.1% Adenine (A), and 17.8% Guanine (G).The phylogram constructed for commercially-important epinephelids and Polyprion americanus (Polyprionidae) was based on COI sequences with a sequence of 622 bps.In this analysis, the separation of the species was supported by high bootstrap values (Fig. 1).
The phylogram indicates that the differentiation of the taxa using the genetic sequences was adequate for the separation of the species.The genera Mycteroperca, Hyporthodus, Epinephelus and Cephalopholis formed distinct clades and Polyprion americanus was confirmed as the outgroup.The robustness of the phylogram is also confirmed by the identification of E. quinquefasciatus as the sister group of E. itajara.
Different size of the fragments of DNA generated using the species-specific primers were analyzed after the electrophoretic reaction, which showed the formation of different bands for each species located in different positions of the gel.The positive control of the reaction was indicated by the F1 and R1 primers of the COI gene, which has approximately 655 bps (Fig. 2).The results indicated that the samples of the different species were amplified by their species-specific primers when applied together in the multiplex PCR.No false positives were found when samples of other fish species were analyzed, indicating only the amplification of the band of the positive control of the COI gene.Fragment sizes: 1200Fragment sizes: , 1000, 700, 600, 500, 450, 400, 350, 250, 150, 50 , 700, 600, 500, 450, 400, 350, 250, 150, 50 bp (Electrophoresis: 1% agarose, 100 V, 100 mA, 90 minutes).

Discussion
Formation of specific clades in the congeneric groups (Epinephelus, Hyporthodus and Mycteroperca) based on the COI gene was similar to the phylogenetic arrangement described by Craig & Hastings (2007) in a molecular study of the Epinephelidae.This emphasizes the reliability of the COI mitochondrial marker for the identification of the species of the family Epinephelidae.
The primers developed here generated distinct patterns of amplification in the multiplex PCR, producing electrophoretic bands of different molecular weights for each of the nine grouper species (Epinephelidae).The use of these primers to discriminate the study species permits considerable savings of time and laboratory work (Teletchea, 2009).Species-specific primers provide lowcost, practical molecular tools that may contribute to the reliable identification of commercial species.This tool has the additional advantage, especially in the context of developing countries, of being relatively easily applied (Abercrombie et al., 2005).The molecular techniques currently available have amplified the perspectives for the application of basic genetic tools to the conservation and management of endangered species in these nations (Bestervan der Merwe & Gledhill, 2015).
Despite being a protected species in Brazil, Epinephelus itajara is subject to illegal commercial fishing along the coast.While the local fishermen are aware of the law prohibiting the exploitation of a number of species, the lack of any effective regulation or controls by government authorities probably encourages illegal fishing (Alexandre de-Franco et al., 2012).The reliable regulation and management of fisheries depends on the adequate identification of the target species and their byproducts.The incorrect identification of fishery products raises a number of concerns, from both economic (fraud) and environmental perspectives, i.e., the management of stocks, due to the reduction in public confidence in the system responsible for the regulation of the fishery industry, and the prevention of the illegal exploitation of protected species (Ogden 2008;Melo Palmeira et al., 2013).In this context, genetic markers capable of discriminating E. itajara represent a valuable tool for the regulation of the fishery industry, and the establishment of a reliable system for the application of the legislation prohibiting the commercial exploitation of this species.
Species-specific primers tool available from this study can also ensure the collection of reliable fishery statistics, which are necessary for the development of effective management and regulatory measures, as well as the conservation of overfished and endangered species (Baker, 2008;Alacs et al., 2010).This question is especially relevant in Brazil, where fishery statistics are largely unreliable and limit the understanding of the commercial exploitation of epinephelids.In fact, official reports often lump together species of the genera Epinephelus, Hyporthodus, and Mycteroperca, and may even include members of other families, such as Polyprion americanus (Bloch & Schneider, 1801), even though this polyprionid is classified as endangered in the IUCN Red List (2015).The inaccurate identification of catches and the marketing of counterfeit fishery products represent a serious threat to the conservation of commercially-valuable fishes such as the groupers, i.e., the Epinephelidae (Asensio, 2008;Carvalho et al., 2015).
The impact of illegal fishing on the Atlantic goliath grouper is of increasing concern due to the distribution of the species in coastal habitats that suffer intensifying anthropogenic pressure, as well as the intrinsic characteristics of the species, such as its longevity, slow sexual maturation, and in particular its habit of forming breeding aggregations that facilitate fishery operations (Sadovy & Eklund, 1999;Sadovy de Mitcheson et al., 2013).However, the traditional tools for the control and regulation of fisheries are extremely limited, and the modern methods available for the identification of epinephelid species, such as real-time PCR (Chen et al., 2012;Ulrich et al., 2013) and DNA barcoding which, while effective, demand expensive equipment and laboratory resources.In addition, no molecular identification procedure is currently in use for the detection of the illegal fishing of the Atlantic goliath grouper in Brazil.
Molecular markers used in the multiplex fingerprinting technique for the authentication, control, and inspection of fishery stocks are based on the design of specific oligonucleotides and markers derived from the sequencebased amplification of the 5S, 12S, 18S (Asensio, 2008, Asensio et al., 2009), 16S (Ulrich et al., 2013), Cyt b (Melo Palmeira et al., 2013), and COI genes (Mendonça et al., 2010), as well as fragment length polymorphisms, RFLPs (Paine et al. 2007;Torres et al. 2013) or even nuclear markers, such as ITS 2 (Feldheim et al., 2010), SNPs (Torres et al., 2013) and microsatellites (Rodrigues et al., 2011), and realtime PCR techniques (Trotta et al., 2005).The COI gene is a highly effective and reliable tool for the diagnosis of the authenticity of fishery products (Veneza et al., 2014), and the multiplex PCR technique has proven especially effective for the detection of endangered species (Mendonça et al., 2010;De-Franco et al., 2010;Alexandre de-Franco et al., 2012).In this case, the low cost of the synthesis of the specific primers for the epinephelid species developed in the present study, and the simplicity of the multiplex PCR technique may favor the adoption of this more effective method for forensic identification, with the added advantage of providing more precise statistics on the fishery exploitation of the epinephelids along the Brazilian coast.
The diversity of endangered and over-exploited fishes, and the extremely restricted laws that protect them, demand the routine use of forensic genetics by the environmental protection agencies in South America (Barletta et al., 2010).The identification of commercial grouper species using relatively simple, low-cost techniques represents an important contribution to the work of the government organs responsible for the regulation of the fishery industry in tropical and subtropical countries of the western Atlantic.
The adoption of the routine molecular differentiation of grouper species (Epinephelidae) using the multiplex PCR of the COI gene developed may be especially effective for the systematic control, regulation, and management of the commercial fishing of these species, due to its low cost, the rapid acquisition of results, and the relatively simple laboratory facilities needed.As endangered species are rarely found in the samples, they are difficult to detect, thereby increasing the costs of processing a large number of samples with potentially negative results.In this case, the approach may be more appropriate for the initial phases of an investigation, with other methods (PCR-RFLP, real-time PCR, genotyping or sequencing) being used to diagnose the samples more specifically during the subsequent phases of the inspection.This should optimize operating costs and time, and provide greater precision in the analysis of the genetic parameters.

Fig. 1 .
Fig. 1.Phylogram based on the amplification of the sequence of the Cytochrome Oxidase I gene of the commerciallyexploited species of fishes of the families Epinephelidae and Polyprion americanus (Polyprionidae).The samples collected in the present study are underlined and those obtained from the GenBank database appear together with their accession numbers.