© 2005 International Council for the Exploration of the Sea
Genetic identification of hake and megrim eggs in formaldehyde-fixed plankton samples
a Departamento de Biología Funcional, Universidad de Oviedo C/Julian Claveria, s/n. 33006-Oviedo, Spain
b Fundación AZTI, Departamento de Recursos Pesqueros Herrera kaia portualdea z/g, 20110 Pasaia (Guipúzcoa), Spain
*Correspondence to E. Garcia-Vazquez: tel: +34 985 102 726; fax: +34 985 103 534. e-mail: egv{at}fq.uniovi.es; palvarez{at}pas.azti.es.
Spawning of European hake (Merluccius merluccius) and megrim (Lepidorhombus whiffiagonis and L. boscii) overlap in time and space. Their eggs are morphologically similar, so genetic identification is needed for purposes of stock assessment based on plankton surveys. Amplification fragment sizes of a partial sequence within the 16S rRNA genes are different for Merluccius merluccius, Lepidorhombus boscii, and L. whiffiagonis. Species-specificity of the pattern was confirmed after analysing adult individuals from all distribution areas of the three species. After Chelex-based DNA extraction and PCR amplification, fragment sizes at this gene were successfully determined from 85% formaldehyde-fixed eggs and larvae recovered from plankton samples. Species-specificity was resolved using an ABI genetic analyser. The results were 100% reproducible. This methodology for genetic identification of hake and megrim eggs can be very useful for stock assessment of these three commercially important fish species.
Keywords: genetic markers, Lepidorhombus boscii, Lepidorhombus whiffiagonis, Merluccius merluccius, mitochondrial genes
Received 24 January 2005; accepted 5 March 2005.
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Introduction |
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Declines of demersal predatory fish due to overfishing have been reported recently (Myers and Worm, 2003). A scientifically-based conservative management is needed to avoid a collapse of the commercial fisheries and the potential extinction of many exploited species. Amongst the many fish species subjected to fisheries, European hake (Merluccius merluccius) and megrim (Lepidorhombus boscii and L. whiffiagonis) are very common in European markets, particularly in southern Europe (Alheit and Pitcher, 1995).
A major concern for managers is the assessment of stock sizes for these three species. For example, uncertainties in age determination may invalidate the use of catch-at-age analytical models for estimating hake stock sizes (ICES, 1996). An alternative way to estimate stock sizes and forecast adult abundance of target fish species is the Egg Production Method. This method is based on the correct identification of eggs collected in plankton surveys. Hake and megrim spawning areas partially overlap from the Northwest Irish coast to the Bay of Biscay, and in the Mediterranean Sea. The three species spawn during the winter, so eggs of all species can be present simultaneously in plankton samples from these areas. Visual identification of fish eggs is possible based on differential morphological characteristics (Moser et al., 1984). However, different species may share identical egg morphology, so identification using morphological characters is better applied to the categories of family or genus only, not to species (Shao et al., 2002). In southern European waters, egg characteristics for these three species such as a lack of differential pigmentation in early stages and morphological characteristics such as identical egg size and oil globule make identification difficult. Therefore, a methodology for genetic identification of the eggs for the three species is needed before the Egg Production Method can be employed for stock assessment. Molecular sequencing is an alternative way to assign an egg to a species as well as to verify the correctness of previous morphological identification (Shao et al., 2002).
Species-specific molecular markers should be highly conserved at the intraspecific level. Many species-specific markers have been developed for purposes of fraud detection in processed fish food (Ram et al., 1996; Carrera et al., 2000; Castillo et al., 2003). Mitochondrial sequences are target regions in the search for species-specific molecular markers for different freshwater and marine species (Medeiros-Bergen et al., 1995; DeSalle and Birstein, 1996; Palumbi and Cipriano, 1998; Lindstrom, 1999; Taylor et al., 2002). The mitochondrial genome is inherited maternally. Many copies are present in eggs even in very early stages. Thus, mitochondrial DNA sequences can be considered the first choice for developing species-specific markers when the aim is to identify fish eggs.
Stock biomass estimates by the Egg Production Method require the processing of several thousands of eggs. If species-specific markers are based on differential fragment size between species, PCR products can be easily screened employing an automated or semi-automated methodology in a sequencer machine, simply labelling the primers with a fluorescent dye. The goal of this study is to develop a methodology for species identification of European hake (Merluccius merluccius) and megrim (Lepidorhombus boscii and L. whiffiagonis) eggs, employing a mitochondrial locus (16S rDNA) as a genetic marker. The universality of the marker was tested in adult samples obtained from different regions of the geographical distribution of each species. Finally, the accuracy of the method was assayed on formaldehyde-preserved eggs obtained in real plankton surveys.
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Material |
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Tissue samples (gill or muscle) from adults caught in different areas of the distribution of the three species were obtained and ethanol-preserved for further analysis. These were used to assess the universality of the molecular marker. The origin and number of the individuals sampled are in Table 1.
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Megrim (Lepidorhombus whiffiagonis) eggs, obtained by artificial fertilization of mature males and females caught in the Bay of Biscay (ICES Area VIIIa 58°39'N–8°08'WIIc) on a research cruise, were used as a control. Gametes were obtained from eight adult males and females collected by bottom trawls in depths of 350–370 m. Oocytes were released by the female following a gentle abdominal massage. Male and female gametes were mixed in a Petri dish with some clean seawater. After a few minutes they were transferred to a 10 l bucket filled with seawater. This moment is considered the time of egg activation and the beginning of ontogenesis. Eggs were fixed 24 h after fertilization. Some eggs were preserved in absolute ethanol, others were preserved in 4% formaldehyde buffered with sodium tetraborate. We analysed genetically 50 eggs from the ethanol-fixed and formaldehyde-fixed samples, respectively.
Bongo nets (60 cm diameter, 303-µm-mesh nets), towed to a nominal depth of 200 m and retrieved obliquely, were used to collect ichthyoplankton samples. Hake eggs at each tow were identified by flotation characteristics (Marrale et al., 1996) and counted before the ship departed from the sampling station, then fixed in 4% formaldehyde solution. This sampling was carried out in ICES Area VIIIc (Bay of Biscay) in 2002. A set of 38 eggs was identified as hake and labelled as WH. A set of larvae was visually identified as megrim, 12 as Lepidorhombus boscii and 31 as L. whiffiagonis. All these eggs and larvae were then fixed in 4% formaldehyde for genetic identification.
Samples of eggs previously identified visually as hake had been obtained from 1995 Celtic Sea (ICES Area VIIj) plankton surveys. They had been preserved in 4% formaldehyde. Some of these eggs were also analysed to test the efficacy of the marker in archived samples. Genetic results were employed to determine the efficiency of visual identification of hake eggs from plankton surveys.
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Methods |
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DNA extraction
DNA was extracted from ethanol-preserved adult samples following the Chelex-based protocol described by Estoup et al. (1996).
DNA was extracted from individual eggs (ethanol- or formaldehyde-preserved) as following. Each individual egg was placed in an Eppendorf tube, rinsed in PBS for 2–3 min, gently squashed with a pipette tip, placed in 150 µl of 12% Chelex + 20 µl proteinase K (400 U/ml) at 55°C for 1 h, then at 100°C for 20 min. The supernatant contains DNA for direct PCR amplification.
PCR protocols and sequencing
PCR amplification of the 16S rRNA mitochondrial DNA segment was performed using the primers H3080 (5'-CCGGTCTGAACTCAGATCACGT-3') and L2510 (5'-CGCCTGTTTATCAAAAACAT-3') described in Palumbi et al. (1991). The reaction was carried out in a total volume of 20 µl including 1.5 mM MgCl2, 0.25 mM dNTPs, 20 pmol of each primer, 20 ng of template DNA, and 1 U of DNA Taq polymerase (Promega). The thermocycler conditions for the amplification were the following: an initial denaturing step at 95°C for 5 min, then 30 cycles of annealing at 95°C for 20 s, 48°C for 20 s, and 72°C for 20 s, and one extension at 72°C for 7 min.
The amplified segment was sequenced for 20 of each species caught in at least three different areas (Bay of Biscay, ICES Area IX, ICES Area VII; at least five of each species per area). The products of the sequencing reactions were visualized in an automated sequencer ABI Prism 3100 (Applied Biosystems) following standard methodology.
Based on the sequences of the three species, two primers were designed for amplification of a sequence fragment different in size between species but conserved within species (primers described in Results). The amplification reaction with these primers is as follows. The reaction mixture was composed of 2 mM MgCl2, 10 pmol of each primer, 0.75 U Taq polymerase (Biotools), 125 µM of each deoxynucleotide in a final volume of 20 µl. The PCR conditions were: 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 45°C, and 30 s at 72°C, and a final extension at 72°C for 10 min.
The product of the PCR amplification with the new designed primers was sequenced for 20 of each species in order to confirm the specificity of the amplified fragment. Sequencing followed the method described above.
Visualization of the PCR products
The fragment sizes of the PCR products obtained with the new set of primers for all sampled individuals (adults, eggs, and larvae) were identified employing an automated ABI Prism 3100 sequencer (Applied Biosystems). One of the primers (16S-A) was fluorescently labelled with HEX. PCR product (1 µl) was mixed with 10 µl of deionized formamide and 0.3 µl of GeneScan-500 ROX Size Standard (Applied Biosystems). Fluorescent fragment detection was performed by capillary electrophoresis in a 3100 Genetic Analyser (Applied Biosystems) with a 36 cm capillary and POP 4 polymer. Electrophoresis conditions were those given as default by the manufacturer. Fragment sizes were established using the GeneScan 3.7 Analysis Software (Applied Biosystems).
All PCR amplifications and fragment size determinations were repeated twice in all formaldehyde-fixed samples analysed, to be sure that the results were reproducible.
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Results |
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The sequences of the 16S rRNA genes obtained after PCR amplification with the universal primers (Palumbi et al., 1991) were variable within species for single nucleotide mutations (SNP). There were also some indels within species, owing to small 1–4 bp insertion/deletions in a few individuals. The most common sequences for each species are shown in Table 2. Based on these sequences we designed a new set of primers for amplification of a shorter sequence fragment. This fragment was different in size between species but conserved within species. The primers, called 16S-A and 16S-B, are marked in grey in the sequence presented in Table 2. These primers are:
- 16S-A: 5'-TGTCTTCGGTTGGGGCGA-3'
- 16S-B: 5'-GCTGTTATCCCTGGGGTAAC-3'
- 16S-B: 5'-GCTGTTATCCCTGGGGTAAC-3'
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The primer 16S-B presented a 1 bp mismatch with both Merluccius merluccius and Lepidorhombus whiffiagonis. However, the fragment amplified was the expected, because it was verified in 20 of each species by direct sequencing. Employing these primers, a partial 16S rRNA gene sequence was amplified in 88 Lepidorhombus boscii, 84 L. whiffiagonis, and 114 Merluccius merluccius adults from different geographical areas (Table 1). The fragment sizes of the PCR products were identified employing an automated ABI Prism 3100 sequencer (Applied Biosystems). The sizes were the following (Figure 1): (i) 154–156 bp for Merluccius merluccius; (ii) 165–168 bp for Lepidorhombus whiffiagonis; (iii) 171–174 bp for Lepidorhombus boscii.
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These fragment sizes were always consistent with those expected from the 16S rRNA sequences presented in Table 2. Some intraspecific variation was found for the three species. However, there was no overlap between species, allowing for unambiguous identification of the three species.
PCR amplification employing the primers 16S-A and 16S-B was successful in all control megrim eggs, both ethanol- and formaldehyde-preserved. The fragment sizes obtained ranged from 165 to 168, identical to that obtained for control adult samples.
Table 3 summarizes the results obtained for the genetic identification of formaldehyde-fixed hake-like eggs and megrim-like larvae obtained from plankton surveys in 1995 and in 2002. Successful PCR amplification and fragment size determinations were achieved for an average of 85% of the samples. In 1995, 87.7% eggs sampled were successfully genetically identified, demonstrating that this method is also useful for long-term stored samples. With respect to species identification, results obtained by visual inspection of the specimens and by genetic methodologies were not identical. On average, 32.7% of the samples were misidentified when species identification was based on morphological traits. Eggs not correctly identified visually were genetically determined in some cases as megrim, because the fragment size obtained with the primers 16S-A and 16S-B was 166 or 167 bp, corresponding to Lepidorhombus whiffiagonis. However, other unknown patterns of fragment size were also found for some eggs (for example 160 bp; two or three fragments of different sizes, and others). On the other hand, larvae visually identified as megrim larvae in 2002 provided 155 bp long fragment sizes, typical of hake. The degree of accuracy of the visual identification of eggs and larvae varied greatly among samples, ranging from 0% in those larvae to 100% in some hake eggs sampled in 1995 (RV "Lough Foyle" and RV "Tridens").
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Discussion |
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The first relevant result of this study was the successful PCR amplification of a mitochondrial DNA segment obtained from formaldehyde-fixed eggs. The method described here allowed identification of 85% formaldehyde-fixed specimens based on fragment size visualization. The reproducibility was 100%, demonstrating that the protocol for DNA extraction employed in this study is adequate for a reliable and reproducible amplification of mitochondrial sequences in formaldehyde-preserved tissues. Our protocol provides improved amplification success with respect to that employed by France and Kocher (1996) and Chase et al. (1998) for rDNA genes in different species. Optimal results for PCRable DNA extraction from archival formalin-fixed tissue based on Chelex methodology have also been reported in the context of human pathological studies (Coombs et al., 1999), probably because single-tube extraction reduces the possibility of contamination. Kirby and Reid (2001) reported successful amplification of the 16S rDNA in a single formaldehyde-fixed larval sandeel. The protocol described in the present study extends the success to many individuals and can be considered for extensive routine surveys at a population level. This result is of particular interest for scientists and managers working on marine species, because it permits analysis of plankton specimens and larvae normally preserved in formaldehyde.
Of relevance is the fact that the fragment sizes obtained for the three species at the 16S rDNA sequence are species-specific for purposes of egg identification. The same pattern was obtained within species for a large number of adults, 88–114 per species. Similar or smaller sample sizes (22–52 sequenced per species) have been used for describing species-specific markers with the same purpose of egg identification in other species such as cod, whiting, and haddock (Taylor et al., 2002). Therefore, the marker described in the present study may be considered universal for identification of hake and megrim.
From our results, misidentification of eggs and larvae can occur when based only on visual inspection. Some samples were correctly identified (Lough Foyle and Tridens in 1995 plankton surveys). However, the degree of uncertainty found for some other samples was very high (RV "Walther Herwig" in 2002, and RV "Heinke" in 1995). Misidentification of larvae based on their morphological traits was also found. Many factors could contribute to a wrong identification of plankton specimens by visual traits. For example, a change of formaldehyde buffering could allow modifications in shape or colour. Some deterioration of specimens by handling during sampling is also expected if sea conditions are unfavourable.
In conclusion, amplification fragments obtained with the primers 16S-A and 16S-B within the 16S rDNA gene provide a species-specific marker for unambiguous identification of formaldehyde-fixed hake and megrim eggs. The use of this marker for plankton studies may be considered by managers charged with ensuring conservation and managing exploitation of marine fish species.
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Acknowledgements |
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This work was supported by the EU Contract MARINEGGS QLK5-CT1999-01157. We are grateful to Ivan Gonzalez Pola (University of Oviedo) for technical support in laboratory tasks.
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References |
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