Food Control 46 (2014) 441e445

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

DNA barcoding reveals a high level of mislabeling in Egyptian sh

llets
Asmaa Galal-Khallaf a, b, Alba Ardura a, Khaled Mohammed-Geba a, b, Yaisel J. Borrell a,
Eva Garcia-Vazquez a, *
a
Departamento de Biologia Funcional, Universidad de Oviedo, C/Julian Claveria s/n., 33006 Oviedo, Spain
b
Genetic Engineering and Molecular Biology Division, Department of Zoology, Faculty of Science, Menoua University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Accurate identication of seafood species in the markets is a growing concern due to the high incidence
Received 23 April 2014 of species substitution at international level. It is a prime priority for governments to be able to identify
Received in revised form the already processed sh products (sh llets). In this context, DNA barcoding was applied to ascer-
6 June 2014
taining species in sh llets (tilapia, Nile perch and panga) purchased from Egyptian markets. Ninety
Accepted 11 June 2014
Available online 18 June 2014
commercial samples were analyzed. Sequencing of a short fragment of mitochondrial cytochrome oxi-
dase I (COI) gene revealed 33.3% species substitution in the sh llets analyzed, 50% Nile perch (Lates
niloticus) and 50% basa sh (Pangasius bocourti) being replaced by imported Vietnamese tra sh (Pan-
Keywords:
COI
gasianodon hypophthalmus). These results demonstrate that DNA barcoding is a reliable tool for detecting
DNA barcoding sh products adulteration in Egypt. We recommend its use for control and law enforcement of seafood
Egyptian food control quality.
Fish llets 2014 Elsevier Ltd. All rights reserved.
Mislabeling

1. Introduction identication (Bottero & Dalmasso, 2011). However, consumers are

Nowadays, the fraudulent mislabeling of sh products has
become an important problem in the seafood industry. Mislabeling
occurs when one species is substituted for another (Rasmussen &
Morrissey, 2008), and it has been revealed worldwide in many
groups of sh (Table 1). Detrimental effects of species substitution
in seafood could be classied in two main types, namely economic
fraud and health hazards (e.g. Galimberti et al., 2013), the cause of
mislabeling being usually the nancial incentive (Von der Heyden,
Barendse, Seebregts, & Matthee, 2010). Major economic deception
or fraud in the seafood industry has been revealed when a less
expensive species is substituted for a more expensive species
(Rasmussen & Morrissey, 2008). To put an end to these practices,
governments have different national and international regulations
for the control of production and preservation of food, as for
example the European directive EC/178/2002 and many others.
Reliable identication of sh species is therefore essential for
detecting and combating mislabeling in markets. Morphological
characteristics represent the rst and obvious line for sh
* Corresponding author. Tel.: 34 985102726; fax: 34 985103534.
E-mail address: egv@uniovi.es (E. Garcia-Vazquez).
not able to identify the sh species when diagnostic traits are ab-
sent, such as in the case of sh llets and other processed food. In
these cases alternative tools are needed for food surveillance. DNA-
based methodologies are especially useful for this purpose because
DNA can be obtained even from highly processed food (Teletchea,
Maudet, & Ha nni, 2005). DNA barcoding is one of the most
widely used and effective methods in food traceability. It is based
on the sequence diversity of a short DNA region (DNA barcode) in
the genome that possesses a high interspecic, but low intraspe-
cic, variability for reliable differentiation between species
(Galimberti et al., 2013). Several markers have already been used
for sh authentication (Wen et al., 2010). The usage of the mito-
chondrial gene cytochrome oxidase I (COI) was rst suggested by
Hebert, Cywinska, Ball, and deWaard (2003) as the core of a global
bioidentication system for animals. The COI barcode region
(around 650 base pair (bp)) is relatively conserved between species
but at the same time shows sufcient variation to allow species
differentiation (Rasmussen Hellberg & Morrissey, 2011). It is
noteworthy mentioning that cytochrome oxidase I (COI) has
become an effective barcode (Hajibabaei et al., 2006) widely used in
various accurate sh and shellsh identication studies, such as
cyprinids, groupers, salmon, crabs, cusk-eels, lutjanids and snake-
heads (Chen, Hsieh, & Hwang, 2012; Chiu, Su, Pai, & Chang, 2012;

http://dx.doi.org/10.1016/j.foodcont.2014.06.016
0956-7135/ 2014 Elsevier Ltd. All rights reserved.
442 A. Galal-Khallaf et al. / Food Control 46 (2014) 441e445

Table 1 frequently chilled or frozen (Namulema, Muyonga, & Kaaya, 1999).

Examples of previous studies showing cases of species substitution in international Finally, panga sh (Family: Pangasiidae, Order: Siluriformes) is a
seafood markets.
Vietnamese catsh cultured in the Mekong River Delta. Almost all
Species on Substitute Reference of the catsh farmed in the Mekong Delta are globally exported to
the label species more than 100 countries, including Egypt, the European Union, USA
Red Snapper Other snappers Food and Drug Administration (2013) and Russia (Globesh, 2010). In recent years, Vietnamese catsh
Red Snapper Rocksh Food and Drug Administration (2013) varieties farmed in that region have been concentrated on the
Mahi Mahi Yellowtail Food and Drug Administration (2013)
genus Pangasius: the basa (Pangasius bocourti), and the tra (Pan-
Swordsh Mako Shark Food and Drug Administration (2013)
Orange Roughy Oreo Dory or Food and Drug Administration (2013) gasianodon hypophthalmus) (Men, Thanh, Hirata, & Yamasaki,
John Dory 2005). They are exported in several forms, mainly as frozen and
Cod Alaska Pollock Food and Drug Administration (2013) thawed llets (Thi et al., 2013). They have become affordable white
Halibut Sea Bass Food and Drug Administration (2013)
sh substitutes for cod and other white eshed shes on the
Dover Sole Arrowtooth Food and Drug Administration (2013)
Flounder
Western market (Noseda et al., 2012). However, Pangasius export
Red Drum Black Drum Food and Drug Administration (2013) value decreased by 7.5% during 2008e9, and its demand uctuates
Yellow Perch White Perch Food and Drug Administration (2013) due to doubts about quality and safety of some sh lots (Hansen &
Yellow Perch Zander Food and Drug Administration (2013) Trifkovic, 2014).
Sturgeon caviar Paddlesh Food and Drug Administration (2013)
In Egypt, the standards for chilled sh (ES: 3494/2005) are
Walleye Sauger Food and Drug Administration (2013)
Atlantic Salmon Pacic Salmon Food and Drug Administration (2013) determined by the Egyptian Organization for Standardization and
Chum Salmon Pink Salmon Food and Drug Administration (2013) Quality Control, as the supreme responsible authority for legalizing
Scallops Skate Wings Food and Drug Administration (2013) food safety parameters. According to the directive ES: 3494/2005, a
Walleye Alaskan Pollock Food and Drug Administration (2013) clear label must be added to all sh products, including the taxo-
Salmon Steelhead Trout Food and Drug Administration (2013)
Wild Caught Farm Raised Food and Drug Administration (2013)
nomic names for species and the family (subject 3/7/3). The level of
Salmon Salmon accomplishment of such normative, however, has not been inves-
Monksh Puffersh Cohen et al. (2009) tigated until now. The aims of this study are two folds. First, the
Longtail tuna Bluen tuna Maralit et al. (2013) cheap, easy and reliable Barcode authentication method has been
Mediterranean Nile tilapia Filonzi et al. (2010)
developed and tested for assisting the authorities to ameliorate
grouper
Gurnard Nile perch Filonzi et al. (2010) food surveillance methods, in particular for controlling the wide
Halibut Tra sh van Leeuwen et al. (2009) international market of frozen sh llets. Second, we have used
Grouper Nile perch Asensio et al. (2009) such DNA barcoding for the detection and quantication of mis-
labeling in commercial raw sh llets purchased from Egyptian
markets. Food inspection applying DNA barcoding, to our knowl-
Cline, 2012; Haye, Segovia, Vera, Gallardo, & Gallardo-Esca rate, edge, is applied for the rst time in the Egyptian markets.
2012; Santaclara, Pe rez-Martn, & Sotelo, 2014; Veneza et al.,
2014; Zhu, Fu, Wang, & Li, 2013).
Fish is an essential food source all over the world. It is about to 2. Materials and methods
be the main alternative source of animal protein (Tidwell & Allan,
2001). In Egypt, sh play an important role in the country's food 2.1. Sample collection
security and domestic economy (FAO, 2003). Frozen sh llets are
one of the most popular products. In the Egyptian markets, they Samples of sh llets labeled as Nile perch (L. niloticus), panga
belong principally to three species/genera: tilapia (Oreochromis (Pangasius or Pangasionodon spp.) and tilapia (Oreochromis spp.)
spp.), Nile perch (Lates niloticus) and panga (Pangasius or Pan- were collected, between September and November 2013, from local
gasionodon spp.). Tilapia (Family: Cichlidae, Order: Perciformes) is markets in three Egyptian governorates; Cairo, Monua and
the generic name of four different species farmed in Egypt. It has Qalyubia. Samples details are shown in Table 2. Names of the
become the dominant Egyptian aquaculture sh, with more than product companies are not disclosed in the study. A small amount
55% of the total aquaculture harvest in 2009 according to the (100 mg approximately) of muscle tissue was xed in absolute
Egyptian General Authority for Fish Resources Development ethanol and stored at 4  C until transport to Spain for further
(GAFRD, 2010). It is the most consumed freshwater sh species in analysis.
Egypt. On the other hand, Nile perch (Family: Latidae, Order: Per-
ciformes), like tilapia, is a warm water sh species. According to 2.2. DNA extraction
FAO (2014), Nile perch is widespread throughout the Ethiopian
Region of Africa, occurring commonly in all major river basins In the laboratory of natural resources of the Department of
including the Nile, Chad, Senegal, Volta and Zaire. It is the most Functional Biology (University of Oviedo, Spain), genomic DNA was
important commercial sh species in East Africa (Muyonga, Cole, & extracted from sh samples preserved in absolute ethanol by the
Duodu, 2004), and its aquaculture is highly valuable and a subject DNA extraction protocol described by Estoup, Largiader, Perrot, and
of innovation (e.g. Hassan et al., 2013). Its export status is most Chourrout (1996) using Chelex resin (Bio-Rad Laboratories). A

Table 2
Number of samples obtained for each species, information stated on the label and collection sites.

Number of samples On label Origin of product Sampling location (province)

Common name (expected species) Processed sh product

20 Nile perch (Lates niloticus) Frozen Fillets Egypt Monua and Qalyubia, Egypt
20 Tra sh (Pangasius hypophthalamus) Frozen Fillets Vietnam Cairo, Egypt
20 Basa (Pangasius bocourti) Frozen Fillets Vietnam Monua, Egypt
30 Tilapia (Oreochromis spp.) Frozen Fillets Egypt Monua, Cairo and Qalyubia, Egypt
 A. Galal-Khallaf et al. / Food Control 46 (2014) 441e445 443

small amount of tissue (approx. 1 mg) was placed in an Eppendorf
tube containing 500 mL of Chelex resin (10%) combined with 7 mL of
proteinase K (400 U mL1). The tissue was incubated at 55  C for
90 min to release DNA. For inactivation of Proteinase K, the tubes
were transferred to 100  C for 20 min. Finally, aliquots of DNA were
stored at 4  C for analysis, and the rest was frozen at 20  C for long
term storage.
www.ncbi.nlm.nih.gov/genbank/), and in the BOLD (www.barcodi
nglife.com) database. Cut-off values for % identity > 95% and
alignment value E 0 were used for identication at species level.
All DNA sequences were aligned using the Clustal W alignment
explorer integrated in MEGA version 5.0 (Tamura et al., 2011). The
haplotypes were determined using DNasp5 program (Librado &
Rozas, 2009).

2.3. PCR amplication 3. Results

Mitochondrial COI partial fragments were amplied by poly-
merase chain reaction (PCR) using the universal primers described
by Ward, Zemlak, Innes, Last, and Hebert (2005). A 50 mL reaction
mixture was prepared containing Promega (Madison, WI) 1 PCR
buffer, 0.2 mM dNTP's, 3 mM MgCl2, 0.5 mM of each primer, 20 ng of
template DNA and 1 U of Taq DNA polymerase. The mixture was run
in a thermal cycler (Applied Biosystems, model 2720) with the
following PCR program: 95  C initial denaturation for 5 min; fol-
lowed by 35 cycles of denaturation at 95  C for 20 s, primer
annealing at 56  C for 20 s and extension at 72  C for 30 s; and a
nal extension step at 72  C for 20 min. After the reaction, the
amplicons were separated by horizontal gel electrophoresis on a 2%
agarose gel stained with 2.5 mL of 10 mg mL1 ethidium bromide.
Amplicons were sent for sequencing in Macrogen Inc. (the
Netherlands), using the Sanger sequencing method.
2.4. Species identication
All amplied sequences were manually corrected using
Chromas lite 2.1.1 software to trim the sequence ends. For species
identication, the revised sequences were compared to reference
sequences in the GenBank database using BLAST algorithm (http://
For the information stated on the labels, the majority of the
samples analyzed in this study (77%) did not include the scientic
name. All of them stated the common names; therefore it was
possible to know the expected species (for Nile perch and panga) or
genus (for tilapia) of each llet (Table 2).
Total genomic DNA was successfully isolated from the 90 sh
llet samples analyzed. The COI primers generated PCR products
that after trimming resulted in size lengths of 610 (Nile perch), 604
(panga) and nally 625 bp (tilapia) for the obtained sequences. To-
tals of 4, 6 and 10 different haplotypes were obtained for Nile perch,
panga and tilapia respectively. These sequences' haplotypes were
submitted to the GenBank database using the Bankit tool and Bar-
code submission option (https://www.ncbi.nlm.nih.gov/WebSub/?
toolgenbank). Their accession numbers are shown in Table 3.
Samples were all identiable by COI sequencing because our
results indicated that all the species examined showed a unique
sequence clearly distinguishable from the others. After comparison
with reference sequences from databases, a high level of mis-
labeling was detected in the frozen sh llets analyzed (Table 3 and
Fig. 1). The identied species did not match with the one declared
on the labels in 50% Nile perch and 50% panga samples analyzed,
given a total 33.3% mislabeling in the analyzed llets. Nile perch

Table 3
Genetic species identication of the analyzed Egyptian sh llets samples. Species name on the label, expected scientic name, Accession Number in the GenBank of the
haplotype obtained for these llets, frequency of each haplotype (%) over the total number of sequences obtained for each sh group, closest match (species and accession
number) in GenBank and BOLD databases, mislabeling (yes/no).

Expected species Accession % Closest match (Accession number in parenthesis) Mislabelling

number
GenBank BOLD

Nile perch
Lates niloticus KJ443710 31.3 Lates niloticus (DQ108019.1) Lates niloticus (DQ108015) NO
Lates niloticus KJ443711 6.3 Lates niloticus (DQ108019.1) Lates niloticus (DQ108015) NO
Lates niloticus KJ443712 6.3 Lates niloticus (DQ108019.1) Lates niloticus (DQ108015) NO
Lates niloticus KJ443713 6.3 Lates niloticus (DQ108019.1) Lates niloticus (DQ108015) NO
Lates niloticus KJ443704 12.5 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (JF292409) YES
Lates niloticus KJ443705 37.5 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) YES
Panga sh
Pangasianodon hypophthalmus KJ443706 32.3 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) NO
Pangasianodon hypophthalmus KJ443707 16.1 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) NO
Pangasianodon hypophthalmus KJ443708 3.2 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (JF292409) NO
Pangasianodon hypophthalmus KJ443709 6.5 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) NO
Pangasius bocourti KJ443705 25.8 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) YES
Pangasius bocourti KJ443707 3.2 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (JF292409) YES
Pangasius bocourti KJ443704 3.2 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (JF292409) YES
Pangasius bocourti KJ443708 6.5 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (JF292409) YES
Pangasius bocourti KJ443709 6.5 Pangasianodon hypophthalmus (JF292405.1) Pangasianodon hypophthalmus (EF609427) YES
Tilapia
Oreochromis spp. KJ443694 52 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (FJ348115) NO
Oreochromis spp. KJ443695 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443700 8 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443696 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443697 8 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443698 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443699 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443701 8 Oreochromis niloticus (GU477625.1) Oreochromis niloticus NO
(GU370126)
Oreochromis spp. KJ443702 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
Oreochromis spp. KJ443703 4 Oreochromis niloticus (GU477625.1) Oreochromis niloticus (GU370126) NO
444 A. Galal-Khallaf et al. / Food Control 46 (2014) 441e445
Fig. 1. Graphical summary of the mislabeling of commercial sh llets in Egyptian markets found in this study. The gure shows the species name on the label (left) and the real
species identied from DNA (right). Percentage of mislabeled products for each case is inserted in the arrow.
substitute species was tra sh (P. hypophthalmus). For panga, basa
sh (P. bocourti) were 100% substituted by tra sh (P. hypo-
phthalmus). These substitutions, especially for Nile perch, were
likely deliberate because the species are morphologically very
different (Fig. 1). On the other hand, mislabeling was not detected
in tilapia samples since all tilapia llets were genetically identied
as Oreochromis niloticus, the local Nile tilapia. Genetics therefore
helped to ascertain the species in 100% cases of sh llets.
4. Discussion
This work represents the rst study using DNA barcoding for
sh species identication in Egyptian markets, focused on the main
species that are sold in Egypt as llets (Nile perch, panga sh and
Nile tilapia). Our results revealed a high incidence of mislabeling. In
essence, 50% of the highly valued Nile perch L. niloticus, was
substituted with the tra sh P. hypophthalmus. Similarly, 100% of
basa sh P. bocourti was also replaced by tra sh. Tra sh has been
reported as a substitute species in other cases, for example for
halibut (Table 1; van Leeuwen et al., 2009). The reason for this
mislabeling could be economic in both cases. Nile perch is more
expensive than tra sh according to FAO Globesh European price
report (2013), and compared to tra, basa have higher production
costs due to higher costs of ngerlings, longer growing time and
higher level of feed consumption (Thanh, 2003). This mislabeling is
thus a clear example of a species of lower price sold as a more
expensive and valuable species, and represents a case of commer-
cial fraud (Filonzi et al., 2010). Moreover, such substitution not only
impinges on Egyptian economy but may have also implications for
health when panga is sold as Nile perch, since panga is farmed in
Mekong River waters (Asia) reported to contain pollutants (van
Leeuwen et al., 2009). Food safety and public health may be at
risk if this practice of seafood substitution becomes generalized.
Mislabeling does not only refer to species substitution. As
mentioned above, Egyptian law enforces a clear and complete
labeling of the frozen sh products, including the scientic name
on labels. However, the labels of the majority of our samples (77%)
did not include the scientic name, representing an infraction of
the Egyptian law ES: 3494/2005. As in other international markets
where DNA-based methodology has revealed seafood mislabeling
(e.g. Cutarelli et al., 2014; Di Pinto et al., 2013; Maralit et al., 2013;
Von der Heyden et al., 2010), the results found in this study conrm
that species substitution is a generalized practice worldwide and
adds Egypt to the long list of countries where it happens. Appli-
cation of reliable methods such as those based on DNA analysis
seems to be increasingly important for ensuring consumers' con-
dence and helping to control food quality (Asensio, Gonz alez,
Rojas, Garca, & Martn, 2009; Hsieh et al., 2010; Triantafyllidis
et al., 2010). The development of molecular and immunological
techniques for species identication started in 1980. These tech-
niques have been largely unused because of their low specicity
and unsuitability in case of highly processed food products
involving chilling, salting and heating (Bottero & Dalmasso, 2011).
In recent years, DNA barcoding has become effective and widely
used in many sh species identication studies, from sardine
(Jerome, Lemaire, Verrez-Bagnis, & Etienne, 2003) to grouper
(Asensio et al., 2009), tuna (Vin ~ as & Tudela, 2009), and cod
(Cutarelli et al., 2014), to name a few. The results found here
conrm its utility also in Nile perch, panga and tilapia. Therefore,
we strongly suggest the application of the COI barcode for sh llet
identication in the Egyptian markets as a high delity tool for
species identication. Lastly, we also suggest that the Egyptian state
agencies should monitor regularly seafood products to impose
more strict controls for product labeling.
5. Conclusion
For the rst time, we provide a tool for detecting mislabeling in
the Egyptian seafood market: DNA barcoding is using COI gene
sequencing as a simple, easy, low cost and authoritative method to
 A. Galal-Khallaf et al. / Food Control 46 (2014) 441e445
identify sh llet products. Since high levels of mislabeling have
been detected (50% for the Nile perch and panga llets analyzed),
we recommend this method for validating label information con-
tents and protecting consumers from health hazards and
fraudulence.
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