© 2005 International Council for the Exploration of the Sea
A double DNA approach for identifying Macrorhamphosus scolopax (Pisces, Centriscidae)
Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki 54 124 Thessaloniki, Macedonia, Greece
*Correspondence to C. Triantaphyllidis: tel: +30 231 0998309; fax: +30 231 0998374. e-mail: triant{at}bio.auth.gr.
DNA-based methodologies are increasingly used successfully to elucidate cases of misidentification of adult individuals of fish species with morphological similarities. However, the problem of misidentification can arise even earlier than adulthood, between congeneric and even distantly related species with similar morphology in the early developmental stages. Therefore, a double DNA approach was developed for Macrorhamphosus scolopax, to identify and discriminate it from European species of the genus Trachurus. These species overlap geographically and temporally and are characterized by morphologically similar eggs. The approach looks at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the 16S rRNA mtDNA gene was followed by restriction analysis with two species-specific enzymes: EcoRV and PmlI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. Additionally, the nuclear multi-copy 5S rRNA gene was selected as an alternative candidate for identifying M. scolopax. The universality of the results was verified by screening a large number of fish from five geographical regions, covering most of the overlapping distribution of the species. The output is a double DNA methodology that can be used for egg identification and which could be of value in the egg production method of biomass assessment.
Keywords: genetic identification, M. scolopax, PCR–RFLP, 5S rRNA, 16S rRNA
Received 29 March 2005; accepted 26 May 2005.
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Introduction |
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Several molecular methodologies for fish species identification have recently been used to circumvent limitations imposed by morphological characterization. These methods have proven of use in preventing unintentional mislabelling of fish, occasionally unravelling cases of commercial fraud (where morphologically similar congeneric species of different commercial importance are mixed and sold as one species), or in estimating stock sizes using the egg production method (where correct identification of eggs to species is crucial; Lockwood et al., 1981).
Many analytical methods have been developed for fish species identification and/or discrimination. Most rely mainly on analysis of proteins, high performance liquid chromatography, and immunoassay (Etienne et al., 2000). However, advances in molecular biological techniques have allowed development of DNA-based methods (Sotelo et al., 1993; Davidson, 1998). Most genetic approaches to the determination of species identity are based on amplification of a region of mitochondrial DNA by Polymerase Chain Reaction (PCR), followed either by direct sequence analysis of the amplified fragment, or restriction fragment length polymorphism analysis (e.g. Carrera et al., 1999). Most DNA analyses for fish species identification have been based on amplification of different mitochondrial DNA regions (Ram et al., 1996; Céspedes et al., 1998). Mitochondrial genes are highly conserved among vertebrates, including fish (Billington and Hebert, 1991), and the inheritance of mtDNA is usually maternal non-recombinational. MtDNA is a broadly used genetic tool, and one of its advantages is the high copy numbers of the mitochondrial genome compared with nuclear genome within a cell. Therefore, mtDNA-based methods can be applied to small amounts of tissue, such as eggs or processed samples. From the two ribosomal RNA (rRNA) genes found in animal mitochondrial genome (12S rRNA and 16S rRNA), the large ribosomal 16S rRNA gene has been used widely for species identification, as well as for phylogenetic analysis (Carrera et al., 1999; Stubbs et al., 2000).
Apart from mtDNA, nuclear genes such as 5S ribosomal DNA (5S rDNA) are possibly suitable candidates for genetic discrimination of related species, because in higher eukaryotes, the 5S rDNA gene comprises a 120-bp highly conserved coding sequence and a variable nontranscribed spacer (NTS). This unit is tandemly repeated, usually arranged head to tail, and is species-specific (Pendas et al., 1994; Belkhiri et al., 1997).
Macrorhamphosus scolopax (a snipe fish, Centriscidae) is widespread in the western Atlantic, western Indian, and Pacific Oceans. Additionally, populations are found in the Mediterranean Sea (Froese and Pauly, 2001; Fish Base online, http://www.fishbase.org). Although the species is not commercially important and information on its biological features is scarce, it overlaps significantly with three species of horse mackerel (Trachurus trachurus, T. mediterraneus, and T. picturatus) in southern European waters (along the coast of the Iberian peninsula and in the Bay of Biscay). As the spawning seasons of these three species overlap with that of M. scolopax, mainly May and June, and they all have similar eggs morphologically (Ré et al., 1982), a genetic methodology is needed to identify/discriminate M. scolopax species from European Trachurus species. Such discrimination methodology will aid accurate identification of eggs.
The objective of this work was to develop simple DNA methods for identifying/discriminating M. scolopax from European Trachurus species using PCR–RFLP analysis of the 16S rRNA mtDNA gene, and a PCR-based agarose gel electrophoresis of the nuclear 5S ribosomal DNA gene. These molecular methodologies can hopefully also be used in future to discriminate other eggs similar to those of horse mackerel.
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Material and methods |
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TopIntroductionMaterial and methodsResults and discussionReferences |
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Samples of M. scolopax were collected from five different regions. In Greek waters, samples were taken off the islands of Chios (central Aegean Sea) and Crete (southern Aegean Sea). Fish specimens were also collected from Palermo (southwest Italy) and Castellan de la Palma (eastern Spain), and from ICES Division IXa (Portugal; Figure 1). White muscle tissue was taken from each fish and stored either at –30°C or in absolute ethanol. Overall, 82 individuals were analysed for 16S rRNA and 87 for 5S rRNA genes (Table 1).
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DNA extraction and PCR amplification conditions
Total genomic DNA was extracted from frozen and ethanol-preserved tissue using the CTAB method described by Hillis et al. (1996). PCR amplification of 16S rRNA segment was performed using the universal primers H3080 and L2510, described by Palumbi et al. (1991). The amplified segment of the 16S rRNA gene had an approximate size of 590 bp. Double-strand DNA amplification was performed in 50 µl reaction volumes containing 1 unit of Taq polymerase (Gibco BRL), 5 µl of 10x Reaction Buffer, 4 mM MgCl2, 0.25 mM dNTPs, 30 pmol of each primer, and approximately 50–100 ng of template DNA.
Thermal cycling amplification conditions were as follows: initial denaturation at 95°C for 4 min, followed by 32 cycles of strand denaturation at 94°C for 1 min, annealing at 50°C for 45 s, primer extension at 72°C for 1 min, and an additional 7-min extension at 72°C. The size of the PCR products was checked against a 100-bp DNA ladder (Gibco BRL) in 1.5% agarose gel, run in 1x TBE buffer and stained with 0.5 µg/µl ethidium bromide.
The set of primers used for the PCR amplification of the 5S rDNA gene was designated as follows: 5SA (5'-TACGCCCGATCTCGTCCGATC-3') (forward primer) and 5SB (5'-CAGGCTGGTATGGCCGTAAGC-3') (reverse primer). These oligonucleotides correspond to primers designed by Pendas et al. (1995) for the amplification of one unit of any tandemly arranged 5S rDNA (including both coding and NTS sequences). The oligonucleotides 5SA and 5SB are designed based on the conserved region of 5S rRNA gene in Oncorhynchus mykiss, and have already been used to amplify a whole unit of the 5S rRNA gene from different fish species (Pendas et al., 1995; Céspedes et al., 1999; Rodriguez et al., 2001), including species of Trachurus (Karaiskou et al., 2003b).
Double-stranded DNA amplification was performed in 25 µl reaction volumes containing 1.5 unit of Taq polymerase (Gibco BRL), 5 µl of 10x Reaction Buffer, 2 mM MgCl2, 0.5 mM dNTPs, 20 pmol of each primer, and approximately 50 ng of template DNA. Thermal cycling amplification conditions were as follows: initial denaturation at 95°C for 4 min, followed by 30 cycles of strand denaturation at 95°C for 20 s, annealing at 62°C for 50 s, primer extension at 72°C for 30 s, and a final 7-min elongation time at 72°C. Electrophoresis of 5 µl of the amplified product was performed for 1 h at 100 V in 1.5% agarose gel, run in 1x TBE buffer, and the gel was stained in a solution containing 0.5 µg/ml ethidium bromide. The size of the PCR products was checked against a 100-bp DNA ladder (Gibco BRL). The resulting DNA fragments were visualized by UV trans-illumination and photographed.
Sequencing of the 16S rRNA gene
Amplified DNA was purified with the Sequenase PCR + Product Sequencing Kit, according to the supplier's protocol. Double-stranded DNA was sequenced manually by dideoxy chain-termination (Sanger et al., 1977) with the Sequenase Kit (Version 2.0, US Biochemical), according to the manufacturer's directions, using primers complementary to the template and [a35S]-dATP (Amersham). Sequencing of the 5' end of the 16S rRNA was carried out using the primer L2510. The sequencing reaction products were electrophorized in a 6% polyacrylamide gel/7 M urea. The gel was dried and visualized by autoradiography on a Kodak film for 3 days.
Endonuclease digestion and restriction fragment length analysis of 16S rRNA
The BioEdit (Hall, 1999) programme was used to search all restriction sites present in 16S rRNA obtained after the sequence analysis of ten M. scolopax. A set of enzymes was chosen on the basis of their predicted patterns, and which, in theory, would allow species identification in comparison with sequences of Trachurus species. Another important requirement was that any restriction enzyme selected should have a target sequence with the lowest intraspecific variability. For this purpose, a large number of fish were screened from various regions in which M. scolopax and the three European Trachurus species overlap. Specifically, 82 M. scolopax and 35 Trachurus spp. were analysed in order to confirm the patterns.
PCR products (5 µl) were subjected to restriction endonuclease digestions with 10 units of each enzyme in a final reaction volume of 10 µl. Incubation temperature and duration of reaction were chosen according to the manufacturer's protocol (New England Biolabs, Hertfordshire, UK). Digested samples were electrophorized in 2% agarose gel using 1x TBE buffer, and stained with 0.5 µg/µl ethidium bromide. The sizes of the resulting DNA fragments were estimated by comparison with a commercial 100-bp DNA ladder (Gibco BRL).
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Results and discussion |
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TopIntroductionMaterial and methodsResults and discussionReferences |
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Sequencing of the 16S rRNA gene
A 590-bp fragment of 16S rRNA gene was amplified successfully in all frozen and ethanol-preserved tissues. The sequence of a 264-bp segment at the 5' end of 16S rRNA was determined for ten M. scolopax (five from the Chios area, two from near Crete, and three from the western Mediterranean). In all, three different haplotypes were determined for M. scolopax, with haplotype M. scolopax1 (Figure 2) shared by all studied populations, and the rest (M. scolopax2 and M. scolopax3) being unique and found only in the Chios sample. The sequences were compared with sequences of 16S rRNA previously obtained for the three Trachurus species (Karaiskou et al., 2003a). In all, 46 polymorphic sites, including eight indels (four insertions and four deletions), were revealed for M. scolopax by reference to the sequence obtained for T. trachurus. Additionally, 41 sites were found capable of discriminating between M. scolopax and the three Trachurus species (Figure 2).
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The transition/transversion ratio falls to almost 3:2 between M. scolopax and the three Trachurus species (22/15). This supports the ratio of transitional mutations to transversions in the mtDNA molecule decreasing towards higher taxonomic levels, because undetectable multiple transitional events at more variable sites accumulate as a function of the time since divergence (Brown et al., 1982).
Identification/discrimination of M. scolopax by RFLP analysis of 16S rRNA
In a search for appropriate endonucleases to distinguish and identify PCR products of M. scolopax from the three Trachurus species, the previous sequences were compared to identify restriction sites within the gene that were reliably different between the species. Two restriction enzymes were potentially useful and inexpensive (Figure 2): EcoRV (GAT
ATC), and PmlI (CACGTG). The sequence comparison revealed one EcoRV/PmlI restriction site present only in M. scolopax, but no restriction site in any of the three Trachurus species.
The results of the RFLP analysis of M. scolopax and the three Trachurus species with the two suitable restriction enzymes are illustrated in Figure 3. For EcoRV digestion, M. scolopax gave a pattern of two fragments of around 340 bp and 250 bp, but there was no recognition site present in any of the Trachurus species. Additionally, for PmlI digestion, M. scolopax gave a pattern of two fragments of around 360 bp and 230 bp, while the PCR product in the three Trachurus species remained uncut, because no recognition site was present. Therefore, the two endonucleases were useful for the discrimination of M. scolopax from Trachurus species.
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As intraspecific variability can undermine the efficiency of the methodology, a large number of fish had to be tested in order to verify the absence of intraspecific polymorphism of the target sequence. This was confirmed by analysing 82 M. scolopax for EcoRV and PmlI, respectively, across the geographical distribution of the species (Table 1). The patterns obtained after screening all samples with the two restriction enzymes showed band sizes that agreed with the expected sizes for the restriction fragments inferred from the sequence analysis, revealing the universality of the PCR–RFLP method, with 100% success.
Several approaches can be followed to identify unknown samples to species. Some studies have compared the sequences of PCR products from samples of taxonomically identified fish to find polymorphic sites with a diagnostic value that could be used to identify unknown samples (Unseld et al., 1995). Quinteiro et al. (1998) proposed approaches that entail genetic distance measurement with phylogenetic tree construction, appropriate for identification of samples belonging to closely related species. However, all the above methods rely on sequencing PCR products, a costly and time-consuming exercise. An alternative and promising approach to sequencing is the analysis of PCR fragments with endonucleases to detect interspecific restriction fragment length polymorphism (Meyer et al., 1995; Ram et al., 1996). The PCR–RFLP of the 16S rRNA gene has been used previously to differentiate between Atlantic salmon and rainbow trout (Carrera et al., 1999), and between other groups of organisms (Riviere et al., 1995; Sato et al., 1998; Jang et al., 2003). The utility of 16S rRNA as a suitable candidate for fish species identification and/or discrimination was also investigated in the present study. The data reinforce the results of previous studies, because digestion of 16S rRNA with specific enzymes and evaluation of the results in a large number of individuals facilitates production of species-specific patterns suitable for discriminating M. scolopax and the three Trachurus species.
Identification/discrimination of M. scolopax species by the 5S rRNA multi-copy gene
The fact that the organization of 5S rDNA offers little intraspecific polymorphism and at the same time a high interspecific variability makes it a very good candidate for identification/discrimination of related species. Interspecific size differences in 5S rDNA are sufficient to distinguish different fish species (Pendas et al., 1995; Céspedes et al., 1999; Karaiskou et al., 2003b).
Based on the utility of this nuclear gene as a tool for species identification, it was selected as an alternative candidate for identification of M. scolopax species. The 5S rRNA gene was amplified successfully, and the size of amplified fragments was checked in agarose gel. As shown in Figure 4, amplification of M. scolopax gave a double band of 350 bp and 370 bp, while the 5S rRNA patterns of the other three Trachurus species, as suggested by Karaiskou et al. (2003b), are totally different, T. trachurus giving a double band of 210 bp and 230 bp, T. mediterraneus a double band of 410 bp and 430 bp, and T. picturatus a pattern of two bands at around 210 bp and 350 bp. The double band present in M. scolopax could be explained as a result of a 20-bp insertion in the spacer (NTS) region of any tandemly repeated unit, or to the presence of a pseudogene.
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As indicated earlier (for the 16S rRNA analysis), the absence of intraspecific variability must be confirmed prior to the choice of a species-specific genetic marker for routine application. Therefore, for the 5S rRNA gene, several individuals were scored, representing the broad geographical range of the species. In all, 87 fish were analysed, covering a large portion of the distribution of M. scolopax. No intraspecific polymorphism was detected, because the pattern obtained and consequently the size of the amplified fragments was the same both within and between populations of the same species. Therefore, 5S rDNA with its interspecific size differences constitutes a very good species-specific nuclear marker, because its PCR amplification is probably the simplest procedure for identifying M. scolopax. The absence of intraspecific polymorphism has also been documented for Trachurus species (Karaiskou et al., 2003b).
The egg production method is based on the accurate identification of eggs collected during plankton surveys. While late developmental stages are easily distinguishable by pigmentation characteristics, and by egg and oil globule size range, such discrimination cannot be used for early stages. As the egg production method uses the earliest developmental stage eggs to estimate the adult biomass, the accuracy of egg identification is considered to be crucial. The present results clearly support the usefulness of two different approaches for discriminating between M. scolopax and three Trachurus species with reliability and confidence, providing a dependable methodology for identifying eggs of these species and hence supporting adult biomass estimation. The PCR–RFLP analysis of the 16S rRNA mtDNA gene provides an alternative, simpler, and faster approach for species identification than sequence analysis, which is expensive and time-consuming. Simultaneously, the combination of the PCR–RFLP analysis with the results obtained by a simple agarose gel electrophoretic analysis of the nuclear 5S rDNA gene could lead to unambiguous identification of M. scolopax from samples containing Trachurus species.
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Acknowledgements |
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We thank N. Kontoudis, N. Tsimenidis, and local fishers for their help with sampling, and P. Lopes, P. Alvarez, and Marco Arculeo for providing samples of M. scolopax. The project is supported by the EU:QLKS-CT 1999-01157 programme under the coordination of E. Garcia-Vazquez (University of Oviedo, Spain). Finally, we thank two anonymous reviewers for their valuable suggestions on an early draft of the manuscript.
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