A rapid and cost effective DNA test is described to identify European eel (Anguilla anguilla) and North American eel (Anguilla rostrata). By means of polymerase chain reaction (PCR) technique parts of the mitochondrial cytochrome b gene are amplified with species specific primers which are designed to produce PCR fragments of different characteristic sizes for European and American eel. The size differences can easily be made visible by agarose gel electrophoresi

Trautner, Jochen

English

Aquatic Commons

Inf. Fischereiforsch. 53, 2006, 49–51 Online first: 19. Juni 2006 6 
© 2006 Bundesforschungsanstalt für Fischerei, Hamburg  DOI: 10.3220/Infn53_49-51_2006 
e-ISSN 1861-2164 
p-ISSN 1860-9902 
Rapid identification of European (Anguilla anguilla) and 
North American eel (Anguilla rostrata) by polymerase 
chain reaction. 
Schnelle Identifikation des Europäischen (Anguilla anguilla) and Nordameri-
kanischen Aals (Anguilla rostrata) mit Hilfe der Polymerasekettenreaktion. 
Jochen Trautner 
Federal Research Centre for Fisheries, Institute of Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany 
jochen.trautner@ifo.bfa-fisch.de 
Abstract 
A rapid and cost effective DNA test is described to identify European eel (Anguilla anguilla) and North American eel (Anguilla 
rostrata). By means of polymerase chain reaction (PCR) technique parts of the mitochondrial cytochrome b gene are amplified 
with species specific primers which are designed to produce PCR fragments of different characteristic sizes for European and 
American eel. The size differences can easily be made visible by agarose gel electrophoresis. 
Kurzfassung 
Es wird ein schneller und kostengünstiger DNA-Test zur Identifikation des Europäischen (Anguilla anguilla) und Nordamerikanischen 
Aals (Anguilla rostrata) beschrieben. Mit Hilfe der Polymerasekettenreaktion(PCR)-Technik werden Teile des mitochondiralen 
Cytochrom-b-Gens mit artspezifischen Primern amplifiziert, die so gewählt sind, dass die entstehenden PCR-Produkte charakte-
ristische Größen für den Europäischen bzw. Amerikanischen Aal aufweisen. Die Größenunterschiede können anschließend leicht 
auf einem Agarosegel sichtbar gemacht werden. 
Introduction 
The population of the European eel (Anguilla anguilla) is 
sharply declining and the recruitment is nowadays at a 
level of 1 % of the level reached in the 60s. In Germany 
efforts are made to restock natural waters like the River 
Elbe with fingerlings in order to support descending 
silver eels hopefully migrating to the Sargasso Sea and 
enhancing the spawning stock capacity in the future. 
In European aquaculture, mainly the European eel 
is used for production. As the price for glass eels has 
risen up to 1000 Euro per kilogram, American glass 
eel (Anguilla rostrata), available for a much lower price, 
is increasingly imported and used as stocking material 
in aquaculture. At fingerling size, it is almost impos-
sible to distinguish between American and European 
eel. As long as those eels are used for the production of 
fish used exclusively for human consumption there is 
no desperate need to ensure species identity. However, 
if aquacultured eels are to be used as fingerlings for 
restocking purposes in European waters it must be 
garanteed that the species is A. anguilla. So the rapid 
identification of those two eel species is an important 
issue concerning the restocking of natural popula-
tions. The consequences of massive stocking with A. 
rostrata in European waters can only be hypothesised. 
Competition with local eels in fresh and costal waters 
for food and habitat is possible as well massive intro-
gression if homing to the Sagrasso Sea is successful. 
There have been different approaches for species dis-
crimination in eel as e. g. protein based methods like 
isoelectric focusing (Rehbein 1998), but also DNA 
based methods like sequencing of PCR fragments 
(Bastrop et al. 2000), restriction fragment length poly-
morphisms (RFLPs) of PCR products (Hwang et al. 
2004; Bastrop et al. 2000), single strand conforma-
tion polymorphisms (SSCP) (Rehbein 1998), and spe-
cies specific primer PCRs (Itoi et al. 2005). 
In this study the mitochondrial cytochrome b gene 
was chosen as a PCR target. As a mitochondrial gene, 
it exists in a high number of copies within the cell. So 
it is easy to amplify mitochondrial DNA fragments 
from all kind of different tissues. Also the nucleotide 
                 
 50 Informationen aus der Fischereiforschung 
sequence is conserved within species so that there is 
not much heterogeneity to be expected within a spe-
cies and the chance of no amplification in the PCR 
due to primer mismatching is low. 
Material and Methods 
We aligned the cytochrome b sequences retrieved 
from Genebank for Anguillla anguilla (AB021776, 
AF006715) Anguilla rostrata (AF006716, AF006717, 
AB021767) and Anguilla japonica (AF006702, 
AF006703, AB021772, AF479272) to design spe-
cific primer pairs for European and American eel 
amplifying parts of the cytochrome b region. 
Using specially designed primers in a 4-primer mixture 
the resulting PCR products for A. anguilla and A. ros-
trata show different sizes, which can easily be distin-
guished by agarose gel electrophoresis: a 789 bp frag-
ment for the European eel amplified with primer pair 
2AngUniCytbF and 5AngAngCytbR and a 589 bp frag-
ment amplified by 1AngRosCytbF and 6AngRosCytbR 
for the American eel. A second amplification product 
for A. rostrata amplified from primers 2AngUniCytbF 
and 6AngRosCytbR with a length of 869 bp is possible 
as 2AngUniCytbF is not specific for A. anguilla but also 
binds in the cytochrome b gene of A. rostrata. 
Primers used: 
1AngRosCytbF:  
5'-CAAAGAAACATGAAACATTGGAGTC-3' 
6AngRosCytbR:  
5'-TTGGTACTACTATTAGAACTAGAATA-3' 
2AngUniCytbF:  
5'-ATGCCCTAGTGGATCTACCAAC-3' 
5AngAngCytbR:  
5'-GCGTAGGCAAATAGAAAATACCAC-3' 
Template DNA was extracted from gill tissue pre-
served in ethanol using 10 % Chelex100 solution ac-
cording to standard protocols. The PCR reaction was 
carried out in a volume of 10 µl using 2.5 mM MgCl2, 
2 mM dNTPs, 1x PCR-Buffer (MWG®), 0.4 U Taq-
Polymerase and 25 pmol of each Primer. Initial de-
natuaration was 3 min, followed by 10 cycles of touch-
down PCR with 40 sec denaturation at 94 °C, 72 °C 
annealing (–1°C each cycle) and 40 sec extension at 72 
°C, followed by 25 cycles at an annealing temperature 
of 61 °C and a final extension of 5 minutes at 72 °C. 
5 µl of the PCR reaction was put on 1% agarose gel 
together with size standards. 
Anguilla rostrata samples from North America and 
Anguilla anguilla samples from the River Elbe were 
used as references in the test. The PCR test was ap-
plied to 200 samples from different aquaculture facili-
ties to identify the species. 
To validate the test and to confirm the identity of the 
PCR products the amplified fragments from the sam-
ples of A. anguilla from the Elbe River and A. rostrata 
from North America were cloned into plasmid vectors 
and sequenced. 
Results 
The results of the PCR reactions on a 1 % agarose 
gel are shown in Figure 1. Lanes 1 and 22 contain a 1 
kb size standard. For the control DNAs of A. rostrata 
(lanes 12 to 15) and A. anguilla (lanes 16 to 19) PCR 
products are visible in the size range of the expected 
length of 789 bp for A. anguilla and 589 bp for A. ros-
trata respectively. 
The negative control with distilled water instead of a 
DNA template did not amplify any DNA (lane 20). 
The analysed samples obtained from eel aquaculture 
facilities (lanes 2 to 11; only 10 out of 200 samples 
shown in Figure 1) show PCR products in a size 
range corresponding to the A. anguilla control reac-
tions (compare lanes 16 to 19) except from one sam-
Figure 1: Agarose gel electrophoreses of species test PCR. Lanes 1 and 22 = 1 kb size standard; lanes 2 to 11 = tested DNA samples; lanes 12 
to 15 = positive A. rostrata controls; lane 16 to 19 = positive A. anguilla controls. lane 20 = negative control ; lane 21 = empty. 
Agarosegelelektrophorese der Arten-Test-PCR. Spuren 1 und 22 = 1-kb-Längenstandard; Spuren 2 bis 11 = getestete DNA-Proben; Spuren 12 
bis 15 = positive A.-rostrata-Kontrollen; Spuren 16 bis 19 = positive A.-anguilla-Kontrollen; Spur 20 = Negativ-Kontrolle; Spur 21 = leer. 
J. Trautner: Rapid identification of European and Northamerican eel by polymerase chain reaction Inf. Fischereiforsch. 53: 49–51, 2006 
                  
51  Informationen aus der Fischereiforschung 
ple (lane 5) that shows a PCR fragment corresponding 
in size to the A. rostrata control (compare lanes 12 
to 15). Additional faint bands are visible in the reac-
tions in lanes 2 to 11 at a size of approximately 200 
bp. Under the described PCR conditions there is only 
very low amplification of the additional fragment (869 
bp) expected for A. rostrata samples by amplification 
of primer 2AngUniCytbF and 1AngRosCytbR (lanes 
12 to 15). 
Cloning and sequencing of the amplified PCR frag-
ments of expected size from the control reactions for 
A. anguilla (lanes 16 to 19) and A. rostrata (lanes 12 to 
15) confirmed the PCR product as the desired part of 
the mitochondrial cytochrome b gene. To gain further 
evidence that the one sample showing a PCR fragment 
corresponding in size to A. rostrata (lane 5)actually 
is  A. rostrata, the fragment was also cloned and se-
quenced together with the PCR product of lane 2, dis-
playing the size of the A. anguilla controls. The result-
ing sequence data clearly identified the sample on lane 
5 as A. rostrata and the one on lane 1 as A. anguilla. 
Discussion 
The presented test is fast, cost effective and reliable 
to discriminate between the European eel A. anguilla 
and the American eel A. rostrata. The time to perform 
the entire test, from tissue to agarose gel photography, 
is approximately 4.5 h, depending on the numbers of 
samples analysed simultaneously. The cost for DNA 
extraction from tissue and the ingredients of the PCR 
reaction are low. 
The additional bands visible in Figure 1 do not inter-
fere negatively with the accuracy of the test, neither 
does the additional band amplified faintly for A. ros-
trata. However, to ensure the reliability of the test, it 
is important to always include positive controls for A. 
anguilla and A. rostrata to the samples analysed. 
Using an approach with a mixture of two primer pairs 
always results in a PCR product no matter which of 
the two species is used as template DNA. Identification 
by positive signals for the two species ensures that a 
failure in the PCR due to e. g. inappropriate reaction 
conditions is clearly indicated by no amplification. If 
the PCR is working properly there is always an am-
plification and a PCR product corresponding to the 
expected length of 789 bp for the European eel and 
589 bp for the American eel respectively. 
To detect other Anguilla species than the described two 
the test could be modified by adding specific primers. 
Considering the fact, that A. rostrata was found in 
samples from aquacultured eel offered for restocking, 
it is highly recommended to ensure species identity of 
a sub-sample by performing DNA tests like the test 
presented here. 
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J. Trautner: Rapid identification of European and Northamerican eel by polymerase chain reaction Inf. Fischereiforsch. 53: 49–51, 2006


Rapid identification of European (Anguilla anguilla) and

North American eel (Anguilla rostrata) by polymerase

chain reaction.

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Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction.

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