GENETIC TRACEABILITY OF SELECTED POPULATIONS OF THE YELLOWSTRIPE SCAD, SELAROIDES LEPTOLEPIS (ACTINOPTERYGII: PERCIFORMES: CARANGIDAE), BASED ON THE ANALYSIS OF MICROSATELLITE DNA—CELFISH PROJECT—PART 3

Background. Monitoring the genetic diversity of fi sh populations can provide information necessary to determine fi shing quotas for the commercially caught species. One of the species subjected to intensive exploitation is the yellowstripe scad, Selaroides leptolepis (Cuvier, 1833), commonly occurring in the coastal waters of Vietnam, Cambodia, Malaysia, and Indonesia. This paper is the third part of a larger project: ”Development of a genetic-based system for identifi cation of food products from fi sheries and aquaculture introduced to the European Union customs area”. Materials and methods. Fin fragments of the yellowstripe scad, Selaroides leptolepis, were obtained in 2012 and 2013 from local markets in Vietnam (SVN), Cambodia (SKH), Malaysia (MMY), and Indonesia (MID). The analyses focused on the amplifi cation and sequencing of a fragment of the nuclear rhodopsin gene which utilized as identifi cation label. Subsequently, in the obtained SVN, SKH, MMY, and MID samples, 11 microsatellite regions (SRR) were analysed. Sequence analyses were performed using the BioEdit and BLAST software, while the analysis of the obtained SRR data was conducted using the GenAlEx software. Results. The analysis of the obtained loci variants divided the investigated populations into two genetically homogeneous groups: the Vietnamese–Indonesian group and the Malaysian–Cambodian group. The analysis of genetic diversity revealed statistically signifi cant deviations from the Hardy–Weinberg equilibrium in the investigated populations and indicated the Vietnamese population as the most stable, while the Malaysian population as the least stable. Conclusions. The study provides information on the genetic diversity of the investigated populations and allows assignment of the captured fi sh to the geographical regions specifi ed. Moreover, indicate that among analysed populations the safest populations were those caught in the local fi sheries (continental shelf) of Vietnam and Cambodia due to their high mean genetic diversity.


INTRODUCTION
Genetic identifi cation of food ingredients is an important method that enables the traceability and confi rmation of authenticity of fi sh products. With regard to common cases in which valuable fi sh species are replaced by low-value equivalents, it is necessary to provide reliable methods which would precisely identify those fi sh species that are valuable for the economy. These methods help to eliminate counterfeit products from markets but also provide tools that might be used by governmental units which control food safety, including custom services. Population identifi cation using molecular methods also provides information about catch region (Anonymous 2015a), which is highly useful in protection strategies of overfi shed populations, or catches of the IUU group (Illegal, Unreported, and Unregulated).
The results presented in this paper are part of the work carried out within the scientifi c research project "Development of a genetic-based system for identifi cation of food products form fi sheries and aquaculture introduced to the European Union customs area" funded by the European Union within 2011-2013. Species of the family Carangidae are the fi fth most intensively exploited fi sh group, following gadoids (Gadidae) (Anonymous 2015b). The yellowstripe scad, Selaroides leptolepis (Cuvier, 1833), is a marine pelagic species occurring in the waters of the Indian Ocean and Pacifi c, from the Persian Gulf to the Philippines, northern Japan, as well as the south coast of the Arafura Sea and Australia. The maximum body size is 22 cm and the weight does not exceed 625 g (Froese and Pauly 2014). The species is the only representative of the genus Selaroides, (family Carangidae), that feeds mainly on mussels and snails, which classifi es the species as predatory. In terms of human consumption, it is a species of an average taste value of the meat, consumed mainly as dried fi sh or used for the preparation of stuffi ng for burgers, but also sold locally as whole fi sh, either fresh or frozen. Studies have shown that due to the high content of red meat combined with the high content of fat and mioglobin, the species is used for the production of high-quality surimi in Asia (Arfat and Benjakul 2012).
Selaroides leptolepis is known in Malaysia and Indonesia under the local name of "kuning" and its market price is within the average values for fi sh for human consumption (Sumaila et al. 2007). In the European Union, the yellowstripe scad was imported as part of the scad fi sh group without a clear indication of a particular species. Within 5 years, there were 57 reports of this species mainly from New Zealand and Sri Lanka (Skrzypczak unpublished). Global catches of the species in 2011 were approximately 200 000 t, while in 2012 the value decreased to 198 600 t (Anonymous 2015b).The natural resources of the yellowstripe scad are most intensively exploited in Malaysia (41 341 t) and United Arab Emirates (3196 t). According to Tan and Yap (2006), the species is the third most intensively captured species after Rastrelliger kanagurta (Cuvier, 1816) and Decapterus maruadsi (Temminck et Schlegel, 1843). It is also reported as trawling bycatch in Australia (Dell et al. 2009). Among the nets used for fi shing are mainly seines, trawls, trapping devices, and nets with a mesh size of up to 20 mm (typically 15 mm). The species status according to IUCN is not evaluated, i.e., not assessed according to the IUCN criteria. The population of that species is rather poorly characterized which is probably due to underestimation of the nutritional value of the S. leptolepis meat, as well as the limited activities for the protection of stocks in countries such as Indonesia, Malaysia, and Vietnam, where the species constitutes native ichthyofauna. These countries are mainly focused on aquaculture, whereas the commonly fi shed wild species are not a subject of population studies. In the Philippines, the yellowstripe scad is defi ned as one of the most often caught species, apart from Decapterus macrosoma Bleeker, 1851; Selaroides leptolepis (Cuvier, 1833); and Rastrelliger kanagurta, although a population analysis allowed the conclusion that the species is exploited too heavily (Rueca et al. 2009). According to Tandon (1964) "overfi shing of marine species and ineffective reintroduction necessitate scientifi c research aimed at species identifi cation, a well as the spatial and temporal modes of spreading of ichthyofauna stocks." The economic signifi cance of the species is high as it is inexpensive and although it is captured using nets with too large mesh (no selectivity of fi shing), it still covers the bulk of the protein demand of local populations. The potential for confusion with other species (Gunn 1990) is primarily due to the morphological similarity and overlapping of the areas of occurrence of 2 species: Selar boops (Cuvier, 1833) and Selar crumenophthalmus (Bloch, 1793). The latter is particularly valued in northern Australia, reaching very high prices. Visual identifi cation of such products is very diffi cult and often impossible. Past experience and controls carried out by various institutions indicate that, for a variety of reasons, the declared fi sh species does not always correspond to the contents of the package (Jacquet and Pauly 2008).
The lack of information necessary to assess the genetic stability of the yellowstripe scad, a relevant species from both economic and ecological point of view, convinced the authors to perform genetic analysis of selected populations of the species.

MATERIAL AND METHODS
The study material consisted of fi n sections from juvenile individuals of the yellowstripe scad, Selaroides leptolepis, obtained in 2012 and 2013 from local markets in Phan Thiet, Vietnam (SVN), Phnom Penh, Cambodia (SKH), Malacca, Malaysia (MMY), and Jakarta, Indonesia (MID).
The collected fi n fragments of the SVN (n = 8), SKH (n = 7), MMY (n = 11), and MID (n = 8) populations were dried for transport and then conserved in 5 mL tubes containing silicone medium. DNA isolation from fi n sections was performed using the peqGOLD Tissue DNA Mini Kit (peqlab) following the attached protocol. The qualitative and quantitative assessment of the isolates was carried out by electrophoresis in 1.5% agarose gel followed by spectrophotometric measurements using the NanoDrop 2000 instrument (Thermo Scientifi c). For all investigated samples, PCR amplifi cation of a fragment of the nuclear rhodopsin gene (RH1) was conducted using the primers Rod-F2W and Rod-R4n in accordance with the methodology proposed by Sevilla et al. (2007). Subsequently, in the obtained SVN, SKH, MMY, and MID samples, 11 microsatellite regions (SRR) were analysed: Orla 21-231, Orla 16-32, Orla 2-91, Orla 22-135, Orla 5-131, Orla 12-160, Orla 16-185, Orla 8-113, Orla 9-204, Orla 9-38, and Orla 20-134. The amplifi cation of the regions was conducted in accordance with the methodology used by Gotoh et al. (2013). The standard PCR profi le was modifi ed to that of touchdown PCR: 94°C for 5 min, then 30 cycles of denaturation (94°C, 45 s), primer annealing (62°C, 30 s × 7 and 59°C, 30 s × 27), and elongation (72°C, 20 s), followed by fi nal elongation at 72°C for 7 min. The reaction mix was prepared based on the GoTaq®G2 Hot Start polymerase (Promega), Green Master Mix 12.5 μL, F-primer 0.5 μL (10 pmol · μL -1 ), R-primer 0.5 μL (pmol · μL -1 ), H 2 O DEPC 10.5 μL, DNA 1 μL. All PCR amplifi cations were carried out on a GeneAmp® PCR System 9700 thermocycler (Applied Biosystems) and the results were evaluated by amplicon electrophoresis in 2% agarose gel. Sequencing of the obtained RH1 PCR products was ordered from Genomed, Warsaw, Poland. Analysis of thus obtained sequences was performed using the BioEdit software and BLAST (Altschul et al. 1990, Hall 1999. The separation of SRR fragments was carried out using the SEQ 8000 sequencer (Beckman Coulter), while the analysis of the obtained SRR data was conducted using the GenAlEx software (Peakall and Smouse 2012).

RESULTS
Comparing the nucleotide sequences of the rhodopsin gene from the SVN, SKH, MMY, and MID samples in the BioEdit software revealed no differences between SVN and MID, and between MMY and SKH. Differences were observed between: SVN and SKH, SVN and MMY, MID and SKH, and MID and MMY. The RH1 sequences were compared with those submitted to GenBank using BLAST and it was found that there was no 100% overlap between the compared sequences and those from Gen-Bank. No sequences originating from this species were also found. A preliminary analysis of the SSR markers demonstrated that all investigated loci in the four populations were polymorphic and, therefore, qualifi ed for further analysis. The total number of alleles for the SVN, SKH, MMY, and MID populations was 89, 85, 114, and 70, respectively, while the total number of alleles of the investigated loci was 4-14 (mean 8.09) for the SVN p opulation, 2-10 (mean 7.73) for the SKH population, 2-17 (mean: 10.36) for the MMY population, and 2-9 (mean 6.36) for the MID population. The mean values of allele distribution with the expected heterozygosity (He) marked for all populations are shown in Fig. 1. The observed heterozygosity (Ho) which determines the ratio between the heterozygous genotypes and the total number of genotypes per locus was between 0.36 and 0.91 (mean 0.65) for the SVN population, between 0.33 and 1.00 (mean 0.65) for the SKH population, between 0.00 and 0.95 (mean 0.70) for the MMY population, and between 0.11 and 0.79 (mean 0.63) for the MID population. Genetic diversity (He) was between 0.44 and 0.85 (mean 0.77) for the SVN population, between 0.48 and 0.88 (mean 0.76) for the SKH population, between 0.1 and 0.92 (mean 0.47) for the MMY population, and between 0.11 and 0.82 (mean 0.69) for the MID population (Table 1). There were statistically signifi cant deviations from the Hardy-Weinberg equilibrium: in the SVN population for loci Orla 16-32 and Orla 12-160 at P < 0.001 and P < 0.01, respectively; in the SKH population for loci Orla 9-38, Orla 16-32 and Orla 20-134 at P < 0.01, P < 0.05 and P < 0.001, respectively; in the MMY population for loci Orla 2-91, Orla 12-160, Orla 9-38 and Orla 20-134 at P < 0.001, P < 0.05, P < 0.01, P < 0.001, respectively; in the MID population for loci Orla 12-160 at P < 0.05. Average genetic diversity of the SVN, SKH, MMY, and MID populations of yellowstripe scad, Selaroides leptolepis was presented in the Table 2. The process of assigning randomly selected individuals to the baseline populations, based on the results of the species identifi cation test, correctly indicated the origin of 75% of the analysed samples. The test was carried out using the signifi cance level of 0.05 (Figs. 2 and 3).

DISCUSSION
Selaroides leptolepis is a species commonly occurring in the waters of the Indo-Pacifi c, quite extensively exploited in such countries as Malaysia (Chong et al. 2010) and the Philippines (Bacalso and Wolff 2014). In general, in countries with access to fresh fi sh of this species, they are consumed directly after catching, however, due to their affordable price, they have also been seen in Europe. In Germany, available is a "snack" product that is produced by smoking (E150), salting, and drying fi llets of the yellowstripe scad. The price is 26.39 EUR per kg. However, the quality of the meat and its sanitary and parasitological evaluation pose a problem. According to Agusa et al. (2007), in the fi sh of the family Carangidae, originating from the coastal waters of Malaysia, high concentrations . Questionable is also the quality of the meat from S. leptolepis as feedstock for the production of surimi or other low-processed products commonly consumed in Asian countries. The study by Peng et al. (2011) confi rmed that the species is a host for muscle larvae of Anisakis sp., Contracaecum sp., and Porrocaecum sp. These parasites are particularly dangerous to people consuming raw fi sh )]/2, χ 2 = Chi-squared statistic χ 2 , P = probability of obtaining the observed Chi-squared value (*P <0.05, **P < 0.01, ***P < 0.001). meat as they may cause a life-threatening parasitic disease known as anisakiosis (herring-worm disease, cod-worm disease). Studies indicate that it is not a species whose meat can be considered as of high quality, however, despite poor toxicological parameters, it is one of the main sources of proteins in the countries of Southeast Asia.
In the case of a species so intensively exploited, it is necessary to determine the characteristics of the population and locate safe places for fi shing. This study demonstrated that populations caught in Vietnam and Indonesia, as well as Malaysia and Cambodia, are genetically homologous. Since the Malaysian population is in the worst condition and its consumption is harmful, a temporary restriction of fi shing or additional monitoring should be introduced. The species is described as migratory between freshwater and ocean in both directions, its migrations are cyclic and its migration range is more than 100 km. It appears, however, that the analysis of microsatellite loci confi rms the hypothesis of isolation of schools/populations that do not mix genetically, which may be associated with the spatial or temporal separation of spawning of specifi c schools, or the existence of another mechanism of isolation (Figs. 2 and 3). In turn, the analysis of the mean genetic diversity of the populations, referred to as intraspecifi c variability, revealed the highest stability in the population caught in the coastal waters of Vietnam and the lowest stability in that of the coast of Malaysia. It is probable that the populations of this species are undergoing genetic regression due to overfi shing and poor environmental conditions of the waters in the coastal zone of Indo-Pacifi c countries, especially near metropolitan areas, such as Jakarta, that do not have proper sewer systems. As was observed in this study, among the investigated fi shing locations, the safest populations were those caught in the local fi sheries (continental shelf) of Vietnam and Cambodia due to their high mean genetic diversity. The exploitation of fi sheries in the zone of continental shelf without respecting catch limits or permitted and safe catching methods causes degradation of unique ecosystems, such as the coastal coral reefs in the Philippines, or destabilizes the entire fi sh populations. In the case of Selaroides leptolepis of the Indo-Pacifi c region, there are still strong and genetically stable populations which should be particularly monitored by fi shing law, while populations undergoing genetic degradation or constituting a part of diet dangerous to human health should be subject to separate fi shing regulations and increased attention of veterinary services.