Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market

Accepted Manuscript Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market Too Chin Chin, Adibah Abu Bakar, Danial Hariz Zainal Abidin, Siti Azizah Mohd Nor PII:

S0956-7135(15)30313-3

DOI:

10.1016/j.foodcont.2015.11.042

Reference:

JFCO 4771

To appear in:

Food Control

Received Date: 9 January 2015 Revised Date:

26 November 2015

Accepted Date: 28 November 2015

Please cite this article as: Chin T.C., Bakar A.A., Zainal Abidin D.H. & Mohd Nor S.A., Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market, Food Control (2016), doi: 10.1016/j.foodcont.2015.11.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market.

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Too Chin China, Adibah Abu Bakar a*, Danial Hariz Zainal Abidin a,b and Siti Azizah Mohd Nor a,b a School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia. b Centre for Research Initiatives – Life Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia.

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*Corresponding author. School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia. Tel. : +6046534017; fax: +604656512. E-mail address: [email protected]

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ABSTRACT

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Seafood mislabelling is a global issue following the increasing worldwide seafood trade

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particularly of processed seafood products, as well as a general lack of regulations and

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tight enforcement in some countries. This study is a pioneering seafood forensics survey

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conducted in Malaysia. A total of 62 seafood samples, either raw, frozen or variously

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processed, were collected from commercial sources. Molecular analyses were performed

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by sequencing Full DNA Barcoding (FDB) with target region of ~ 700 bp or Mini

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Barcoding (MDB) with smaller target region of only ~150 bp. The DNA barcode

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sequences obtained were compared with those available on BOLD and GenBank

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databases. The DNA targets were successfully amplified and sequenced from 81% of

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seafood samples. Among these samples, 16% were found to have been mislabelled at

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source. This study supports the view that DNA barcoding can be a powerful tool in

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seafood forensics.

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1.0 Introduction

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Food authentication is increasingly conducted by food safety authorities,

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particularly in many developed countries due to concerns arising from several high

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profile cases of food mislabelling and substitution. For instance these include detection

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of donkey and/or horse meat in salami (Primrose et al., 2010) and beef burgers in United

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Kingdom (O’Mahony, 2013). Seafood is one of the most common protein sources

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consumed worldwide due to its high nutritional value and seemingly endless supply. In

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the case of seafood, fraud usually refers to cases of deliberate species substitution,

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claiming expensive fish for cheaper ones or farmed fish for wild-caught (Armani et al.,

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2011; Armani et al., 2012; Cutarelli et al., 2014). In fact, increasing international trade in

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processed seafood and fairly widespread lack of regulation and enforcement has favoured

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such activities (Hanner et al., 2011; Hellberg & Morrissey, 2011). Seafood mislabelling

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can lead to dire consequences such as the misrepresentation of stock numbers, thereby

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compromising sustainable fisheries. This practice could severely impact conservation

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efforts, particularly of protected or critically endangered species. It could also lead to

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potential health risks for consumers, resulting in the loss of consumer confidence in the

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food supply (Noguchi & Arakawa, 2008; Stiles et al., 2011; Hanner et al., 2011).

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Since the early study by Wong & Hanner (2008) of seafood adulteration in

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Canada and the United States, there have been many similar reports (Warner et al., 2012a,

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Warner et al., 2012b; Warner et al., 2013; Cline, 2012). In particular, an extensive

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investigation conducted from 2010 to 2012 by Oceana, the largest international

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organization which focuses exclusively on ocean conservation, observed that no less than

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33% of 1,215 seafood samples collected from 674 retail outlets in USA were mislabelled

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(Warner et al., 2013). European seafood markets are also subject to high rates of

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mislabelling. Cod products in the United Kingdom and Ireland (Miller et al., 2012; Miller

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& Mariani, 2010), hake in Spain (Machado-Schiaffino et al., 2008) and Greece (Garcia-

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Vazquez et al., 2011), fish and fish products in Italy (Armani et al., 2011; Armani et al.,

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2012; Cutarelli et al., 2014) have all been reported to be mislabelled and/or substituted by

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cheaper species. In Asia, the incidence in seafood mislabelling has been documented in

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Taiwan (Hsieh et al., 2010; Huang et al., 2014), Japan (Iguchi et al., 2013), and the

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Philippines (Maralit et al., 2013). Seafood mislabellings in Latin American countries such

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as Brazil (Palmero et al., 2013), Belize (Cox et al., 2013) and Chile (Haye et al., 2012)

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have been shown to be rampant and also in South Africa (Cawthorn et al., 2012), Taken

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together, these works highlight seafood fraud as a serious global issue. Seafood authentication covers the whole range of seafood products available in

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the markets; from fresh whole fish, fish fillets, molluscs and crustaceans to frozen,

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cooked, battered, deep fried, salted, pickled, smoked, grilled, heavily processed (fish

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balls, crab sticks etc.), canned products, as well as sushi and sashimi (Wong & Hanner,

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2008; Nicolè et al., 2012; Armani et al., 2015; Jérôme et al., 2003). Fillets and processed

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seafood are as might be expected more prone to be mislabelled compared with whole

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fish, as morphological features which might be helpful for identification are absent. In

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addition, processes such as steaming, cooking, roasting and mixing with various sauces

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also lead to DNA degradation in the samples (Armani et al., 2015).

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In recent years advances in molecular technology have made a large contribution

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to efforts to improve food authentication methods. Generally, mitochondrial genes are the

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preferred analytical targets over nuclear genes for the identification of species due to their

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numerous advantages: 1) They are short and evolve rapidly, 2) They do not contain

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introns, pseudogenes and repetitive sequences which might hinder efficient sequence

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alignment, 3) multiple identical copies exist in each cell facilitating DNA amplification

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and sequence recovery from degraded tissue and 4) complete mitogenomes known for

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many organisms and readily accessible in online databases, which enable the design of

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amplification primers both universal and specific types (Mackie et al., 1999; Teletchea et

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al., 2005; Nicolè et al., 2012). The mitochondrial cytochrome b gene (Armani et al.,

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2011; Armani et al., 2012) and 16S rRNA gene (Trotta et al., 2005; Horreo et al., 2013)

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have been widely applied for species identification. However, in the last decade the DNA

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barcoding approach using cytochrome c oxidase subunit 1 (CO I) has taken precedence

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over other methods for identification of seafood products due to its particular

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effectiveness (Wong & Hanner, 2008; Nicolè et al., 2012; Cawthorn et al., 2012;

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Cutarelli et al., 2014). This technique was first introduced by Hebert et al. (2003) and

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facilitates species identification by sequencing this one specific mitochondrial gene. This

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fast, inexpensive and highly reliable laboratory method owes its effectiveness to the

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extensive bank of reference sequences contained in the dedicated Barcode of Life

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Database (BOLD) that includes all the DNA Barcodes produced during the forensic

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studies described above.

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In Malaysia, the administration of food safety comes under the authority of the

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Food Safety and Quality Division (FSQD) at the Ministry of Health (MOH). This single

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authority regulates the national standards for processed foods, agricultural products,

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meat, dairy and fisheries. All food products sold must be in compliance with the

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Malaysian Food Act 1983 and Food Regulations 1985. Among other stipulations they

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require that all food packages are properly labelled with the following information: 1) an

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appropriate designation of the food or a description of the food containing the common

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name(s) of its principal ingredients 2) a declaration of the presence of additives and a

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common name(s) of the animal product 3) name and address of the manufacturer and/or

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packer 4) minimum net weight in grams; 5) a list of ingredients 6) full storage

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instructions 7) the country of origin and any other necessary details.

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Bearing in mind that seafood mislabelling has been extensively reported

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worldwide, it is therefore timely that a study should be conducted to investigate the

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Malaysian scenario, with a view to consumer protection. The aim of this study, that 4

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represents the first attempt of utilizing the DNA barcoding in the seafood sector, was to

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evaluate the incidence of seafood mislabelling on the Malaysian market.

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2.0 Materials and Methods

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2.1 Seafood samples

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Seafood samples were purchased from local supermarkets, chain stores and two sushi

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bars in Penang, Malaysia (see later in Table 2). All products were brought back to The

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School of Biological Sciences, Universiti Sains Malaysia, identified by an internal code,

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photographed and stored (at room temperature, 40C or -200 C, depending on the

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packaging typology) until further analysis.

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2.2 DNA extraction and amplification

Two types of extraction methods were used. The DNA from raw and frozen samples was

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extracted using a high-salt DNA extraction protocol based on that of Aljanabi & Martinez

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(1997). The DNA extraction from processed samples (e.g. canned, salted, fermented,

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marinated, seasoned, sauce, tempura, chips, flavoured balls, nuggets, dumplings, floss,

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satay and cooked or grilled fish) were conducted using the Qiagen DNeasy mericon Food

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Kit (Cat. No: 69514), according to the protocol provided by the manufacturer. Extracted

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DNA was quantified using UV spectrophotometer Q3000 (Quawell, USA). DNA

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concentration was expected to be between 10-200 ng/µl and the purity of DNA in the

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range of 1.8-2.0 ratio of absorbance wavelength A260/A280. Electrophoretic analysis

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was also done using 1% agarose gel to examine the degree of degradation for the

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extracted DNA. For the polymerase chain reactions (PCR) several pairs of primers (as

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shown in Table 1) were tested to amplify CO I regions from successful DNA extracts.

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Basically, the fish samples were tried with F1, R1 and F2, R2 primers, whereas others

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samples were tested with universal LCO, HCO primers. If these primers failed to

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amplify, then combination of F1, R2 primers, as well as VF1_t1, VR1d_t1, which are

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incorporated with M13 primers were used.

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Table 1

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To obtain high throughput DNA sequencing of PCR products, we used primers

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described by Ivanova et al. (2007) and Meusnier et al. (2008) for the obtaining of a Full

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DNA Barcode (FDB) of ~700 bp and a Mini DNA Barcode (MDB) of ~150 bp. Both

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authors suggested designs with incorporated M13 sequencing primers. Reactions were

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performed in 25 µl volumes containing 2.5 µl of Buffer (10x Mg2+ free), 2.0 µl of MgCl2

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(25 mM), 2.0 µl of dNTP (2.5 mM), 0.4 µl of each primer (10 µm), 0.3 µl of Taq DNA

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polymerase (5U/µl), 2.0 µl (50 ng/µl) of DNA template, and 15.4 µl of double distilled

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water (ddH2O). All reagents were obtained from INTRON i-TaqTM Plus (Cat. No.:

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25152). PCR amplifications (except for reactions with universal mini-barcode primers –

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see below) were carried out according to the following regime: initial denaturation at

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94ºC for 2 mins, 35 cycles of denaturation at 94 ºC for 30-45 sec, annealing at optimized

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temperature for 30-45 sec, extension at 72ºC for 30-45 sec, and a final extension at 72ºC

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for 8-10 mins. The touch up PCR protocol edited from Meusnier et al (2008) was as

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follows: 95°C for 2 min, followed by 5 cycles of 95°C for 1 min, 45°C for 1 min, and

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72°C for 30 sec, followed by 35 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for

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30 sec, and finally a long extension at 72°C for 5 min. Successful PCR products were

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sequenced by First Base-Asia Sdn. Bhd. using ABI3730xl Genetic Analyzer (Applied

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Biosystem, Foster City, CA).

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2.3 Sequence Data Analysis

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Bi-directional DNA sequences obtained from each sample were aligned in MEGA

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version 6.0 software (http://www.megasoftware.net/) (Tamura et al., 2013). For species

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identification, amplified sequences were compared with reference sequences entered in

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the BOLD (http://www.boldsystems.org/) and GenBank (http://www.ncbi.nlm.nih.gov)

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databases. Samples were considered identified at species level when there was less than

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1% difference with reference sequences (Ratnasingham and Hebert, 2007). A distance-

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based approach by constructing a Neighbour-Joining tree (Saitou & Nei, 1987), also

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drawn using the MEGA version 6.0 software, was used to display results. Reliability for

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the distance tree clusters was evaluated by a bootstrap test (Felsenstein, 1985) with 10

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000 replications.

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3.0 Results and Discussion

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3.1 Sample collection

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A total of 62 seafood samples were collected for analysis and consisted of both local

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(50%) and imported (50%) brands. Among these, 54 samples were heavily processed

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samples which consist of 13% of surimi–type products and 8 samples were either raw or

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fresh/frozen (Table 2). Majority of the products collected gave only a general description

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on the package but with no information on taxonomic details.

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Table 2

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3.2 Molecular analysis

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Using spectrophotometric analysis, 52 samples (83.9%) produced DNA of

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sufficient quantity and quality for PCR amplification (see Table 2). In fact, 10 samples

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showed poor quality for PCR amplification (absorbance ratio value of less than 1.8-2.0).

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We also run an electrophoretic analysis on all ten of these samples using 1% agarose gel

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to examine their degree of degradation. Low molecular weight smearing was observed in

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each sample lane (confirming their highly degraded condition).

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From the 52 good qualities DNA extracts, 48 (92%) individual targets were

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successfully amplified by FDB as stated in Table 3 while only 2 samples (S8 and S9)

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were amplified by MDB. Surprisingly, templates from samples S24 and S41 failed to

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support PCR amplification using any of the primer pairs despite apparently having good

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quality of DNA and being in sufficient concentration.

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Table 3

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We then began a closer examination of those samples whose DNA extracts had failed to yield PCR Products.

For samples S5 (fish chip surimi) and S36 (crab

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dumpling), we failed to extract good quality of DNA despite their being labelled as

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containing 61% and 65.6% of fish meat respectively. We suspected these samples were

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either to contain low actual amount of fish meat used, intense processing had highly

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degraded the DNA or maybe only essence of fish/crab was utilized in products.

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Meanwhile, among canned seafood products (S7-S13), only samples S8 and S9 were

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successfully amplified by MDB and sequenced. The DNA extracts from the remaining

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five canned seafood samples failed to produce PCR products, most probably owing to

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the low quality of the extracted DNA, as a consequence of the canning process, which

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involves heating, high pressure and sterilization. Extensive exposure to extreme

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conditions during this industrial process tends to degrade DNA into short fragments

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(Cawthorn et al., 2012; Chapela et al., 2007; Lin & Hwang, 2007; Hellberg & Morrissey,

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2011; Teletchea et al., 2005).

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A high amount of gelatinous protein is thought to have caused failure in

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extracting good quality DNA from sample S21 (jellyfish sushi). This type of contaminant

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hampers DNA extraction because the compound binds tightly to nucleic acids during the

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isolation of DNA and interferes with subsequent reactions (Winnepenninckx et al., 1993).

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Maybe a more rigorous extraction procedure is needed to ensure gelatinous substances

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can be removed from jellyfish sample as reported by Armani et al., (2013) which failed to

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be achieved by both of the extraction procedures used in this study.

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Finally, we suspected that the presence of other inhibitors especially high salt

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concentration in sample S43 (‘Budu’-fermented anchovy) and S46 (crabstick) hence 9

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failed to be extracted. ‘Budu’ is a fish sauce and originated from Kelantan, Malaysia. It

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is traditionally made by mixing anchovy and salt which are allowed to ferment for 140 to

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200 days. Crabsticks are a formed from cured surimi combined with additives such

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as monosodium glutamate. Extraction or detection of genomic DNA from refined or

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fermented foods such as ‘Budu’ and crabstick may be near impossible, because of

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elimination or degradation of genomic DNA through manufacturing or fermentation

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processes (Yoshiteru et al. 2006). Moreover, any sort of physical or chemical treatment of

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food samples may result in a decrease in the average genomic DNA fragment size due to

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random cleavage of these macromolecules. Therefore, a more gentle procedure is needed

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to prevent DNA damage, especially during the extraction stage, which sometimes

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involves an excessive mechanical shearing (Marmiroli, 2003).

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Reviewing these results from our study, three types of processed foods were

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difficult to extract DNA. These were ones which involved canning or contain high

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amount of protein or other inhibitors. These items are seemed suffer severe genomic

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DNA fragmentation during their manufacturing process thus failed to be amplified by

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PCR.

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3.3 Genbank and BOLD search

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Among the 50 seafood product samples whose long barcodes were analyzed, 66%

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(33 samples) were easily identified and found to be in more or less perfect agreement

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with the general description or common name on product label. For the remainder 9

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samples have identification discrepancies between BOLD and GenBank databases and 8 10

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samples were confirmed to be mislabelled products. Table 4 shows the list of seafood

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samples and results obtained based on the sequence identification using both BOLD and

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GenBank databases.

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Table 4

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A phylogenetic approach based on the construction of a NJ tree with validated

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reference sequences from BOLD and GenBank was employed to display all the results

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from the similarity searches (Fig. 1). The samples clustered into broad general categories

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of fish, clams, jellyfish, prawn, cuttlefish, octopus and squid. Members of related species

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form single clades. It is noteworthy that all fish-based products with general labels

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surveyed in this study (fish cocktail tempura S4; fish ball S6; fish popcorn S27; seafood

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nugget S31, white fish ball S32; fish sandwich S37) consisted of Alaska Pollock or

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Bigeye (Priacanthus spp.). In addition, the presence of a seafood mixture was detected in

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S23. This product was labelled as prawn cuttlefish ball composed of 56% surimi, 20%

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cuttlefish meat and 5% prawn meat. The fish-based surimi was mixed with cuttlefish

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meat cubes, which allowed the amplification of both fish and cuttlefish barcoding targets

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from using primers VF1_t1 and VR1d_F1, and LCO 1490 and HCO 2198 respectively.

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As the proportion of prawn meat was relatively low, it was not surprising that PCR failed

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to amplify any prawn DNA sequence from this sample.

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Fig. 1

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3.3.1 Database discrepancies

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Nine samples were found to have apparent identification discrepancies between

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the GenBank and BOLD system. Sample S9 was identified as Nematopalaemon tenuipes

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(97.56%) on BOLD, whereas its match in GenBank was Synalpheus stylopleuron (83%).

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Three samples, S25, S32 and S38, which were identified as Crassostrea angulata (99%),

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Ilisha elongate (91%) and Pellona ditchela (90%) respectively on GenBank, did not have

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available matches on BOLD and therefore their taxonomic status could not be

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determined. Our GenBank search detected S28 as Metapenaeus dobsoni (86%), whilst

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BOLD identified the sample as Metapenaeus sp. (99.05%). S56 was recognized as

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Octopus sp. (98.62%) on BOLD but Amphioctopus cf. siamensis (92%) on GenBank. In

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addition, S42 was identified as Rastrelliger kanagurta (100%) on BOLD but as

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Scomberomorus plurilineatus (91%) on GenBank. Both are the species of mackerel. S50

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was identified as Amphioctopus aegina (99.69%) on BOLD but Amphioctopus

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marginatus (99%) on GenBank. The fish finger sample, S3, was identified as Gadus ogac

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(Greenland cod) on GenBank, but as Gadus macrocephalus (Pacific cod) on BOLD.

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According to Carr et al. (1999), the Greenland cod shows basically identical

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mitochondrial sequence with the Pacific cod. The Greenland cod may represents a

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contemporary range extension of the Pacific cod. Therefore, the authors suggest that the

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Greenland cod be synonymised into Pacific cod. This is similar to the case of Alaska

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Pollock and Norwegian Pollock as described above for sample S22.

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In the cases of clear discordance between the two databases, the BOLD

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identification would always be granted higher provisional acceptability as it has been 12

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developed based on verified sequences and tagged specimens (Wong & Hanner, 2008).

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However, the larger GenBank database contains more sequences is needed the

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prospective verification process with material of uncertain provenance. However,

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GenBank does contain a mixture of validated and non-validated sequences and sequence

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errors are not uncommon (Forster, 2003).

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3.3.2 Mislabelling

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Unexpectedly, most of the mislabelled products analyzed in our study were sushi-

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based. Samples S15, S16, S17, S18 and S49 were sushi roe purchased from different

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sushi bars. Samples S15 to S18 were labelled as “ebikko”, which commonly refers to

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prawn roe. By observation, the samples were categorised according to colours although

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not labelled as such; (S15-natural orange and S16- green, from sushi bar A);S17– natural

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orange and S18 – dark, from sushi bar B). Sequencing results showed that all four

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samples were mislabelled. They were in fact capelin or smelt roes, which should be

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labelled as “masago”, identified simply as Mollotus villosus on BOLD but as Mollutus

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villosus villosus on GenBank. According to the Fisheries and Aquaculture Department of

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Food and Agriculture Organization of the United Nations (FAO), the latter is considered

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to be a synonym and Mollotus villosus is accepted as the valid Latin bipartite name of the

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capelin. Studies have documented that capelin roe are also a common substitution for

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flying fish roe (Bledsoe et al., 2003; Wong & Hanner, 2008). Traditional sushi made

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from flying fish roe is called “tobiko” and the term “ikura” and refers to salmon roe. Only

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S49 was labelled correctly as salmon roes. Meanwhile, for sample S22, king crab sushi

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turned out to be Alaska Pollock (Gadus chalcogrammus) based on BOLD or the

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Norwegian Pollock (Theragra finnmarchica) according to GenBank and certainly not the

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succulent crustacean from family Lithodidae. Several studies have shown that the Alaska

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Pollock is synonymous to Norwegian Pollock with the former being introduced from the

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Pacific to the Atlantic Ocean (Ursvik et al., 2007; Byrkjedal et al., 2008). Maybe the

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sushi was actually made from surimi-based products and was made to resemble king crab

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meat through flavouring and shaping. Similarly, we found both snow crab leg (S29) and

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crab meat squid ball (S33), to contain Mauvelip threadfin bream, Nemipterus mesoprion

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and red bigeye (Priacanthus macracanthus) respectively, but no traces of any crab meat.

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We propose that such products should be labelled and/or advertised to show clearly that

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they are imitations.

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In addition, it is perhaps not surprising that sample S8 was labelled as fried

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canned sardines but confirmed to be the island mackerel, Rastrelliger faughni based on

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both BOLD and GenBank databases. It is a fairly common usage among locals to refer to

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canned fish as sardines, perhaps referring to the packing nature of the fish in the

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containers. Thus, it is uncertain whether this mislabelling was an intentional deception or

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due to routine cultural perception.

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3.3.3 Critically endangered species

It is unfortunate to note that our study also detected the use of endangered species

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in seafood products. We found two samples to consist of species which have been listed

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under critically endangered species list by IUCN (Limburg & Waldman, 2009). These are 14

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the European eel, Anguilla anguilla (S19), and the Southern bluefin tuna, Thunnus

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maccoyii (S20). For S20, we made a further examination by comparing this sample

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sequence with all samples used by Lowenstein et al. (2009). This examination is done

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because "Bluefin tuna" can refer to three species, T. thynnus (northern Bluefin tuna), T.

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orientalis (Pacific bluefin tuna), or T. maccoyii (southern Bluefin tuna) which is the one

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that is endangered. We successfully found character-based key to identify southern blue

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fin tuna at position 508.

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Studies showed that the decline of eel fisheries is mostly due to anthropogenic

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factors such as; overfishing, pollution, habitat loss and degradation, dam construction,

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river obstruction, parasitism, as well as global warming. All of these factors reduce

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marine productivity (Bonhommeau et al., 2008a,b;). Therefore, critical conservation

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measures such as restoration of habitat, environmental impact surveys and enforcement

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of fishing and environmental legalities are in immediate need of implementation with

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respect to its declining catch. The southern bluefin tuna is a quota-managed species in

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fisheries (Hartog et al., 2011). Its total allowable catch (TAC) for the years 2012 to 2015

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ranged from 10,449 to 12,449 tonnes (Commission for The Conservation of Southern

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Bluefin Tuna (http://www.ccsbt.org/site/). From the viewpoint of seafood management,

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all products that potentially contain these two species should be forensically monitored

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regularly.

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4.0 Conclusion

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In some countries, taxonomic details (Hanner et al. 2011; Nicole et al., 2012;

343

Maralit et al., 2013; Gallal-Khallaf et al., 2014) or adherence to an accepted fish list or 15

ACCEPTED MANUSCRIPT

trade name of the fish product is a requirement (US Food and Drug Administration and

345

the Canadian Food Inspection Agency are examples of agencies already employing this

346

practice). In contrast, only a common name is required on food labels in Malaysia (Laws

347

of Malaysia Food Act 1983 and Food Regulation 1985 Regulation No 156- 170 for Fish

348

and Fish Products). Less stringent regulations on food labelling may decrease detection

349

of mislabelling, as many species would be classified into a single group and therefore

350

lead to a false assurance that seafood misrepresentation is low in this country. In our

351

view, which is based strictly on present evidence, amendment to the existing Act and

352

Regulation should be seriously considered as using many different, but closely related,

353

species (therefore identified as a single group) may cause unwelcome responses from

354

consumers and raise potential health concerns. Furthermore, in an effort to penetrate the

355

global market, additional compliance with taxonomic information and international

356

legislation is timely to ensure acceptability of domestic foodstuffs for export.

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Overall, the rate of mislabelling has been found to be fairly low in this research

358

project, (16%) compared with other market substitution reports we examined. However,

359

we did manage to have a comparable sample size to other relevant published studies

360

(Wong and Hanner 2008; Hanner et al., 2011; Cawthorn et al., 2012; Nicole et al., 2012;

361

Changizi et al., 2013; Maralit et al., 2013; Warner et al., 2013; Cutarelli et al., 2014;

362

Gallal-Khallaf et al., 2014). Our study has proven the utility of DNA barcoding technique

363

for seafood authentication and surveillance in Malaysia. Its general utilisation could

364

enhance transparency and fair trade in the domestic fisheries market and acceptability in

365

the global market. Seafood fraud is occurring in Malaysia on a scale roughly similar to

366

other countries and this can cause negative impacts on the fisheries, stock management

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16

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and conservation of the endangered species and consumer health. Therefore, we

368

recommend that seafood authentication should be conducted frequently and implemented

369

together with the more stringent enforcement of regulations. The authorities should

370

monitor closely to assure sustainable fishing in Malaysian waters and instil consumer’s

371

rights and raise awareness in the public.

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Acknowledgments

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We thank members of Lab 308, Universiti Sains Malaysia for laboratory assistance and

376

Prof. Chambers for his input and suggestions as well as comments on an earlier draft. We

377

would also like to thank the two reviewers for their insightful comments, which have

378

greatly improved the clarity. This work was supported by APEX Delivering Grant from

379

Universiti Sains Malaysia: 1002/PBIOLOGI/910317.

380 381

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ACCEPTED MANUSCRIPT Table 1. Primer pairs tested to amplify COI region in current study. M13 primers were highlighted.

5’-3’ Sequence

TGTAAAACGACGGCCAGTTCCACTAATCACAARGATATTGGTAC

Reference Folmer et al., 1994 Folmer et al., 1994 Ward et al., 2005 Ward et al., 2005 Ward et al., 2005 Ward et al., 2005 Ivanova et al., 2007 Ivanova et al., 2007 Meusnier et al., 2008

CAGGAAACAGCTATGACGAAAATCATAATGAAGGCATGAGC

Meusnier et al., 2008

GGTCAACAAATCATAAAGATATTGG TAAACTTCAGGGTGACCAAAAAATCA TCAACCAACCACAAAGACATTGGCAC TAGACTTCTGGGTGGCCAAAGAATCA TCGACTAATCATAAAGATATCGGCAC

RI PT

ACTTCAGGGTGACCGAAGAATCAGAA TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG

CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA

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Name LCO 1490 HCO 2198 F1 R1 F2 R2 VF1_t1 VR1d_t1 UniminibarF1_t1 UniminibarR1_t1

ACCEPTED MANUSCRIPT

Label

Main ingredients

Brands

Typology

Extraction

Salmon in mustard sauce Tempura salmon goujon Cod fish finger Fish cocktail tempura Fish chip Fish ball

Salmon 55% Salmon 55% Cod 55% White fish 55% Surimi 62% Surimi 80%

Imported Imported Imported Imported Local Local

Processed Processed Processed Processed Processed Processed

S7 S8

Canned fried mackerel in chilli sauce Canned fried sardines with chilli

Mackerel Sardines

Local Local

Processed Processed

Successful Successful Successful Successful Successful Successful Unsuccessful

S9

Canned prawn sambal

Prawn

Local

Processed

Successful

S10

Canned tuna flakes in sunflower oil

Tuna

Local

Processed

Unsuccessful

S11

Canned tuna chunks in water

Tuna

Local

Processed

Unsuccessful

S12

Canned sardines in tomato sauce

Sardines

Local

Processed

Unsuccessful

S13 S14 S15

Canned mackerel in tomato sauce Salmon sushi Sushi roe sushi in natural colour

Mackerel Salmon Not mentioned

Local Imported Imported

Processed Raw Processed

Unsuccessful

S16 S17 S18 S19 S20

Sushi roe sushi in green colour Sushi roe sushi in natural colour Sushi roe in dark colour Eel sushi Tuna sushi

Not mentioned Not mentioned Not mentioned Eel Tuna

Imported Imported Imported Imported Imported

Processed Processed Processed Processed Processed

S21

Jellyfish sushi

Jellyfish

Imported

Raw

Successful Successful Successful Successful Successful Unsuccessful

S22

King crab sushi

Imported

Processed

S23

Prawn cuttlefish ball

Crab meat Surimi 56%, cuttlefish 20%, prawn 5%

Local

Processed

AC C

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Sample ID S1 S2 S3 S4 S5 S6

SC

Table 2. Details on the analyzed commercial seafood products in this study.

Successful

Successful Successful

Successful Successful

ACCEPTED MANUSCRIPT

TE D

Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Processed Raw Processed Processed Processed Processed Processed Processed Processed Raw Processed Processed Processed Frozen

SC

RI PT

Local Local Local Local Local Local Local Local Local Local Local Local Local Local Local Local Imported Local Local Local Local Imported Local Imported Imported Imported Imported Imported Imported Imported Imported Imported Imported Imported Imported Imported

M AN U

Surimi Oyster Squid 70% Alaska Pollock 45% Not mentioned Not mentioned Surimi 71.5% Surimi 71.5% Surimi 71.5% Not mentioned Squid 55% White fish 55% Surimi 65.6% Fish 55% Fish Fish Marlin Oyster extract Mackerel Fermented Anchovy Prawn Salmon Crab stick Salmon Salmon roe Salmon Octopus Yellowtail Eel Jellyfish Bonito Cuttlefish Octopus Mackerel Capelin Mussel

EP

Crab claw Oyster tempura Squid Fish popcorn Tempura prawn Snow crab leg Lobster flavoured ball Seafood nugget White fish ball Crab meat squid ball Squid ring tempura Fish cocktail tempura Crab dumpling seafood roll Fish sandwich Dried fish fillet Fish satay Fried marlin floss Oyster sauce Salted mackerel “Budu” Cooked prawn sushi Flame-grilled salmon belly sushi Deep-fried eggs and shredded crab stick Raw salmon slice sushi Salmon roe sushi Deep-fried salmon skin Cooked octopus and marinated onion slices Yellowtail with onion and mayonnaise sushi Grilled eel sushi Seasoned jellyfish Fried bonito Raw cuttlefish sushi Seasoned baby octopus Grilled mackerel Grilled capelin New Zealand mussel

AC C

S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48 S49 S50 S51 S52 S53 S54 S55 S56 S57 S58 S59

Unsuccessful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful Unsuccessful Successful Successful Successful Successful Successful Successful Unsuccessful Successful Successful Unsuccessful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful Successful

ACCEPTED MANUSCRIPT

Shark Catfish Salmon King salmon

Imported Imported Imported

EP

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SC

RI PT

Fish fillet Atlantic salmon fillet New Zealand king salmon

AC C

S60 S61 S62

Frozen Frozen Frozen

Successful Successful Successful

ACCEPTED MANUSCRIPT Table 3. PCR and sequencing primers for various samples and their respective annealing temperatures applied in this study. Annealing temperature 45ºC

S34

44 ºC

S1, S2, S4, S6, S45

51 ºC

F1/ R1

S47, S54 S49, S60, S62

F2/ R2

S30, S31

S18, S19, S22, S27, S29, S35 S37, S40

50 ºC

48 ºC

51 ºC 50 ºC

S3, S14,S15, S17, S23

51ºC

S8, S9

45 ºC for 5 cycles and 50 ºC for 35 cycles

EP AC C

52 ºC

S48, S51, S52, S57, S58

TE D

VF1_t1/ VR1d_t1 UniminibarF1_t1/ UniminibarR1_t1

48 ºC

M AN U

F1/ R2

RI PT

LCO 1490/ HCO 2198

Samples S16, S23, S25, S26, S28, S32, S33, S38, S39, S42, S44, S50, S53, S55, S56, S59, S60

SC

Name

ACCEPTED MANUSCRIPT

Species identification

Sample ID

S5 S6 S7 S8 S9* S10 S11 S12 S13 S14

Fish cocktail tempura Fish chip Fish ball Canned fried mackerel in chilli sauce Canned fried sardines with chilli Canned prawn sambal Canned tuna flakes in sunflower oil Canned tuna chunks in water Canned sardines in tomato sauce Canned mackerel in tomato sauce Salmon sushi

Priancanthus macracanthus (99.84%) (Red bigeye) Failed to amplify

SC

Cod fish finger

Oncorhynchus gorbuscha (99%) (Pink salmon) Oncorhynchus gorbuscha (99%) (Pink salmon) Gadus ogac (99%) (Greenland cod) Gadus chalcogrammus (99%) (Alaska pollock) Failed to amplify

M AN U

S4

Tempura salmon goujon

Oncorhynchus gorbuscha (100%) (Pink salmon) Oncorhynchus gorbuscha (99.85%) (Pink salmon) Gadus macrocephalus (100%) (Pacific cod) Gadus chalcogrammus (100%) (Alaska pollock) Failed to amplify

Priancanthus macracanthus (99%) (Red bigeye) Failed to amplify

TE D

S3*

Salmon in mustard sauce

GenBank (BLAST)

Rastrelliger faughni (97.33%) (Island mackerel)

Rastrelliger faughni (94%) (Island mackerel)

Nematopalaemon tenuipes (97.56%) (Shrimp) Failed to amplify

Synalpheus stylopleuron (83%) (Shrimp) Failed to amplify

Failed to amplify

Failed to amplify

EP

S2

BOLD

AC C

S1

Label

RI PT

Table 4 BOLD and BLAST results from query sequences that were derived from the analyzed commercial seafood products.

Failed to amplify

Failed to amplify

Failed to amplify

Failed to amplify

Salmo salar (100%) (Atlantic salmon)

Salmo salar (99%) (Atlantic salmon)

ACCEPTED MANUSCRIPT

Sushi roe sushi in natural colour

Mallotus villosus (99.85%) (Capelin)

Mallotus villosus villosus (99%) (Capelin)

S16

Sushi roe sushi in green colour

Mallotus villosus (99.85%) (Capelin)

Mallotus villosus villosus (99%) (Capelin)

S17

Sushi roe sushi in natural colour

Mallotus villosus (99.85%) (Capelin)

Mallotus villosus villosus (100%) (Capelin)

Sushi roe in dark colour

Mallotus villosus (99.85%) (Capelin)

Mallotus villosus villosus (100%) (Capelin)

Anguilla anguilla (99%) (European eel) Thunnus maccoyii (100%) (Southern bluefin tuna)

Anguilla anguilla (99%) (European eel) Thunnus maccoyii (99%) (Southern bluefin tuna)

Jellyfish sushi

S22 King crab sushi S23 Prawn cuttlefish ball S24 S25* S26 S27 S28* S29 S30

Crab claw Oyster tempura Squid Fish popcorn Tempura prawn Snow crab leg Lobster flavoured ball

Failed to amplify

SC

M AN U

S21

Tuna sushi

Failed to amplify

Gadus chalcogrammus (100%) (Alaska pollock)

S23a- Priacanthus tayenus (100%) (Purplespotted bigeye) S23b- Sepia latimanus (99.7%) (Broadclub cuttlefish) Failed to amplify

Theragra finnmarchia (100%) (Norwegian pollock) Gadus chalcogrammus (100%) (Alaska pollock) S23a- Priacanthus tayenus (99%) (Purplespotted bigeye) S23b- Sepia latimanus (99%) (Broadclub cuttlefish) Failed to amplify

TE D

S20

Eel sushi

No match

EP

S19

Ommastrephes bartramii (99.68%) (Neon flying squid) Gadus chalcogrammus (100%) (Alaska pollock) Metapenaeus sp. (99.05%)

Crassostrea angulata (99%) (Portuguese oyster) Ommastrephes bartramii (99%) (Neon flying squid) Gadus chalcogrammus (100%) (Alaska pollock) Metapenaeus dobsoni (86%)

Nemipterus mesoprion (100%) Mauvelip threadfin bream Priancanthus macracanthus (99.84%) (Red bigeye)

Nemipterus mesoprion (100%) Mauvelip threadfin bream Priancanthus macracanthus (99%) (Red bigeye)

AC C

S18

RI PT

S15

ACCEPTED MANUSCRIPT

S36 S37 S38* S39 S40 S41 S42* S43 S44 S45 S46 S47 S48 S49 S50*

Crab dumpling seafood roll Fish sandwich Dried fish fillet Fish satay Fried marlin floss Oyster sauce Salted mackerel “Budu” Cooked prawn sushi Flame-grilled salmon belly sushi Deep-fried eggs and shredded crab stick Raw salmon slice sushi Salmon roe sushi Deep-fried salmon skin Cooked octopus and

Todarodes pacificus (99%) (Japanese flying squid) Gadus chalcogrammus (100%) (Alaska Pollock)

Failed to amplify

Failed to amplify

Gadus chalcogrammus (100%) (Alaska pollock) No match

Gadus chalcogrammus (100%) (Alaska pollock) Pellona ditchela (90%) (Indian pellona) Upeneus margarethae (99%) (Margaretha’s goatfish) Tetrapturus angustirostris (99%) (Shortbill spearfish) Failed to amplify Scomberomorus plurilineatus (91%) (Queen mackerel) Failed to amplify

Upeneus cf. margarethae (99.54%) (Margaretha’s goatfish) Tetrapturus angustirostris (100%) (Shortbill spearfish) Failed to amplify Rastrelliger kanagurta (100%) (Indian mackerel) Failed to amplify

RI PT

Fish cocktail tempura

Todarodes pacificus (99.51%) (Japanese flying squid) Gadus chalcogrammus (100%) (Alaska Pollock)

SC

S35

Squid ring tempura

Priancanthus macracanthus (99.84%) (Red bigeye)

M AN U

S34

Crab meat squid ball

Priancanthus macracanthus (99%) (Red bigeye) Ilisha elongate (91%) (Herring) Priancanthus macracanthus (99%) (Red bigeye)

TE D

S33

White fish ball

Priancanthus macracanthus (99.84%) (Red bigeye) No match

Litopenaeus vannamei (100%) (Pacific white shrimp) Salmo salar (100%) (Atlantic salmon) Failed to amplify

Litopenaeus vannamei (99%) (Pacific white shrimp) Salmo salar (99%) (Atlantic salmon) Failed to amplify

Salmo salar (100%) (Atlantic salmon) Oncorhynchus gorbuscha (100%) (Pink salmon) Salmo salar (100%) (Atlantic salmon) Amphioctopus aegina (99.69%)

Salmo salar (99%) (Atlantic salmon) Oncorhynchus gorbuscha (99%) (Pink salmon) Salmo salar (99%) (Atlantic salmon) Amphioctopus marginatus (99%)

EP

S32*

Seafood nugget

AC C

S31

ACCEPTED MANUSCRIPT

S53 S54 S55 S56*

Seasoned jellyfish Fried bonito Raw cuttlefish sushi Seasoned baby octopus

(Coconut actopus) Seriola quinqueradiata (99%) (Yellowtail) Anguilla rostrata (99%) (American eel) Nemopilema nomurai (99%) (Nomura’s jellyfish) Katsuwonus pelamis (99%) (Bonito) Sepia recurvirostra (99%) (Curvespine cuttlefish)

Octopus sp. (98.62%)

Amphioctopus cf. siamensis (92%)

M AN U

Scomber scombrus (99.85%) Scomber scombrus (99%) (Atlantic mackerel) (Atlantic mackerel) S58 Mallotus villosus (99.7%) Mallotus villosus villosus (99%) Grilled capelin (Capelin) (Capelin) S59 Perna canalicula (100%) Perna canalicula (99%) New Zealand mussel (New Zealand green-lipped mussel) (New Zealand green-lipped mussel) S60 Pangasius hypophthalmus (100%) Pangasius hypophthalmus (98%) Fish fillet (Shark catfish) (Shark catfish) S61 Salmo salar (99.84%) Salmo salar (99%) Atlantic salmon fillet (Atlantic salmon) (Atlantic salmon) S62 Oncorhynchus tshawytscha (100%) Oncorhynchus tshawytscha (99%) New Zealand king salmon (King salmon) (King salmon) Mislabeled products are presented in bold print while samples with * were samples with identification discrepancies between BOLD and BLAST databases.

EP

TE D

Grilled mackerel

AC C

S57

Grilled eel sushi

(Sandbird octopus) Seriola quinqueradiata (100%) (Yellowtail) Anguilla rostrata (99.69%) (American eel) Nemopilema nomurai (99.84%) (Nomura’s jellyfish) Katsuwonus pelamis (100%) (Bonito) Sepia recurvirostra (99.54%) (Curvespine cuttlefish)

RI PT

S52

marinated onion slices Yellowtail with onion and mayonnaise sushi

SC

S51

ACCEPTED MANUSCRIPT 100 79

100

100

100

RI PT

63

77

100

SC

87

100

M AN U

98 100

100 100

100 100

68

100

TE D

100

56

100

EP

65

AC C

S48 Salmon roe sushi Oncorhynchus gorbuscha EF455489.1 Oncorhynchus gorbuscha JX960913.1 S2 Tempura salmon goujon Oncorhynchus gorbuscha JX960912.1 S1 Salmon in mustard sauce S62 New Zealand King salmon Oncorhynchus tshawytscha HQ167683.1 S61 Atlantic salmon fillet S45 Flame-grilled salmon belly sushi S47 Raw salmon slice sushi S49 Deep-fried salmon skin Salmo salar KF792729.1 Salmo salar JQ390056.1 S14 Salmon sushi Salmo salar BT044032.1 S3 Cod fish finger Gadus ogac DQ356941.1 S35 Fish cocktail tempura Gadus chalcogrammus AB182304.1 S4 Fish cocktail tempura Gadus chalcogrammus AB182300.1 S27 Fish popcorn S22 King crab sushi Gadus chalcogrammus AB182301.1 S22 King crab sushi Theragra finnmarchica AM489719.1 S54 Fried bonito Katsuwonus pelamis AB101290.1 S42 Salted mackerel Scomberomorus plurilineatus JF494457.1 S57 Grilled mackerel Scomber scombrus KJ205419.1 S23a Prawn cuttlefish ball Priacanthus tayenus FJ238019.1 S6 Fish ball S30 Lobster flavoured ball S31 Seafood nugget S33 Crab meat squid ball Priacanthus macracanthus JQ681316.1 S20 Tuna sushi Thunnus maccoyii JN086150.1 S60 Fish fillet Pangasianodon hypophthalmus JN021313.1 S58 Grilled capelin S18 Sushi roe (dark color) S15 Sushi roe (natural color) S17 Sushi roe (natural color) Mallotus villosus villosus FJ205579 Mallotus villosus villosus FJ205579.1 S16 Sushi roe (green color) Mallotus villosus villosus FJ205582.1 S39 Fish satay Upeneus margarethae KC147801.1 S8 Canned sardines Rastrelliger faughni KF009654.1 S40 Fried marlin floss Tetrapturus angustirostris HM071007.1 S19 Eel sushi Anguilla anguilla KJ564259.1 S52 Grilled eel sushi Anguilla rostrata KJ564207.1 S29 Snow crab leg Nemipterus mesoprion EF609559.1 S38 Dried fish fillet Pellona ditchela FJ347929.1 S32 White fish ball Ilisha elongata HM030771.1 S51 Yellowtail sushi

100 99

100 100 73 100 72 100 100 90 100

Fish

Fig 1. Neighbour-Joining tree showing the relationship of sample sequences with validated reference available in BOLD and GenBank databases. Samples with red symbol were detected to be mislabelled.

26

ACCEPTED MANUSCRIPT

100 100 100

92 65 83 100

72

76 100 62 95

97 92

M AN U

100 100

97 100

100

AC C

EP

TE D

Fig 1. Continue.

RI PT

98

S51 Yellowtail sushi Fish Seriola quinqueradiata AB517556.1 S59 New Zealand mussel Perna canaliculus AF308731.1 Clams S25 Oyster tempura Crassostrea angulata KC170323 S53 Seasoned jellyfish Jellyfish Nemopilema nomurai JQ353747 S28 Tempura prawn Metapenaeus dobsoni KF453210.1 S44 Cooked prawn sushi Prawn Litopenaeus vannamei AY781297.1 S9 Canned prawn sambal Synalpheus stylopleuron KJ625060.1 S55 Raw cuttlefish sushi Sepia recurvirostra AB430413 Cuttlefish S23b Prawn cuttlefish ball Sepia latimanus voucher KF009663.1 S50 Cooked octopus Amphioctopus marginatus KF489433.1 Octopus S56 Seasoned baby octopus Amphioctopus cf. siamensis AB430516.1 S34 Squid ring tempura Squid Todarodes pacificus AB158364.1 S26 Squid Squid Ommastrephes bartramii AF000057.1

SC

100 100

ACCEPTED MANUSCRIPT Highlights:

AC C

EP

TE D

M AN U

SC

RI PT

• DNA barcoding is an effective tool for seafood authentication. • Seafood authentication should be conducted in larger scale which covers more brands and products around Malaysia. • 16% were found to have been mislabelled