Food Control 130 (2021) 108264

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

Authentication of fish species served in conveyor-belt sushi restaurants in

Taiwan using DNA barcoding
Chia-Hao Chang a, Meng-Ling Tsai b, Ting-Ting Huang a, Yu-Chun Wang c, *
a
TIGP Biodiversity Program, Tunghai University, No.1727, Sec.4, Taiwan Boulevard, Xitun District, Taichung City, 407224, Taiwan
b
Department of Life Science, Tunghai University, No.1727, Sec.4, Taiwan Boulevard, Xitun District, Taichung City, 407224, Taiwan
c
Planning and Information Division, Fisheries Research Institute, No. 199, Hou-Ih Road, Keelung City, 202008, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: Fish fraud is a problem worldwide. Not only does it erode customer confidence, but it also jeopardizes public
Fish fraud health. Typically, it is difficult to authenticate fish species identity based on morphological characters after food
Food processing processing. Fortunately, DNA is relatively resistant to processing methods, and DNA barcoding has proven useful
Mislabeling rate
for species identification. Although fishery product mislabeling is recognized as an issue in Taiwan, a respective
Molecular authentication
Labeling regulations
study on sushi has not yet been conducted. Here, we successfully DNA-barcoded 121 out of 122 sushi samples
from eleven chain restaurants of the conveyor-belt type in Taiwan, revealing an overall mislabeling rate of
~17.36%. We found that capelin roe is commonly used to replace other fish roe, such as herring. Tuna, which is
susceptible to fraud, was always correctly labeled. We also noted inconsistent usage of Chinese common names,
conflict between Chinese and English names, and ambiguous usage of Japanese kanji in Chinese labels, all
serving to confuse species identity. Moreover, spurious reference sequences in the BOLD database and the lack of
a standard list of common names of fishes with their corresponding scientific name both impede investigations of
fish substitution. We advocate that the responsible authorities establish a reliable barcoding database for mo­
lecular authentication of fishes, publish a respective standard for common and scientific names, and legislate for
more comprehensive labeling regulations based on experience in other jurisdictions to facilitate mislabeling
investigations and to deter food fraud.

1. Introduction
The United Nations predicts that the global human population will
reach almost 11 billion by the end of this century (https://population.un
.org/wpp/). Therefore, ensuring sufficient nutrition for everybody is a
crucial issue for all governments. Fish is a major protein source and
according to the FAO Yearbook of Fishery and Aquaculture Statistics
2018, global fish consumption is estimated to be 20.3 kg per capita,
representing ~17.3% of animal protein intake (FAO, 2020).
The strong demand for fisheries products, combined with declining
fish stocks, have resulted in fish becoming one of the most traded food
commodities globally. Fish is generally processed prior to being sold to
extend its shelf-life. However, processes such as filleting can remove
important morphological characters. Moreover, a typical fish supply-
chain can involve multiple countries, with fish being caught (har­
vested or farmed), subjected to primary (e.g. filleting) and secondary
processing (e.g. canning), and then packaged in different countries (Hu,
Huang, Hanner, Levin, & Lu, 2018). The absence of identifying char­
acters, together with supply chain complexity makes fishery products
very vulnerable to food fraud (Pardo, Jiménez, & Pérez-Villarreal,
2016). Numerous fish substitution studies have been conducted, which
reveal that although mislabeling rates can vary according to region, fish
species, and product types, seafood fraud is a common phenomenon
around the world (Calegari, Avila, Reis, & Alho, 2020; P.-Y.; Chen et al.,
2020; Delpiani et al., 2020; Hu et al., 2018; Kappel & Schröder, 2020;
Liou, Banda, Isaacs, & Hellberg, 2020; Mitchell, Rothbart, Frankham,
Johnson, & Neaves, 2019; Pardo & Jiménez, 2020; Sameera, Jose,
Harikrishnan, & Ramachandran, 2021; Shehata, Naaum, Garduño, &
Hanner, 2018; Sultan, Ali, Hossain, AsingNaquiah, & Zaidul, 2018;
Xiong et al., 2018; Xiong, Yuan, Huang, & Xiong, 2020).
Fish species substitution or mislabeling may be unintentional or
deliberate. Unintentional mislabeling occurs when similar fish species
are misidentified, when fish species have different vernacular names in
different regions or distinct fish species share the same vernacular name

* Corresponding author.
E-mail address: ycwang@mail.tfrin.gov.tw (Y.-C. Wang).

https://doi.org/10.1016/j.foodcont.2021.108264
Received 13 February 2021; Received in revised form 11 May 2021; Accepted 19 May 2021
Available online 5 June 2021
0956-7135/© 2021 Elsevier Ltd. All rights reserved.
C.-H. Chang et al.
(Pardo & Jiménez, 2020), or product information is lost along the supply
chain (Cohen et al., 2009). Deliberate fishery product mislabeling may
be driven by financial gain or the desire to gain market access, which can
foster illegal, unreported and unregulated (IUU) fishing (Blanco-­
Fernandez, Garcia-Vazquez, & Machado-Schiaffino, 2021). Overall,
consumer deception, harm to human health, smuggling and illegal
fishing, jeopardizing fisheries management, and religious implications
are all serious consequences of unintentional and deliberate fish mis­
labeling (Barendse et al., 2019; da Silva et al., 2019; Delpiani et al.,
2020; Charlebois, Schwab, Henn, & Huck, 2016; Kroetz et al., 2020;
Kusche & Hanel, 2021; Minoudi et al., 2020; Shehata et al., 2018;
Staffen et al., 2017; Willette et al., 2017; Williams, Hernandez-Jover, &
Shamsi, 2020; Xiong et al., 2018; Yao et al., 2020).
Sushi, a traditional Japanese cuisine, prepared using vinegared rice
and various toppings and fillings, usually raw seafood, is a popular kind
of food worldwide. Trade globalization, healthy eating campaigns, and
ease of preparation have all promoted sushi to being a popular cuisine
globally (Greenberg, 2010; Issenberg, 2008; Staffen et al., 2017). Tai­
wan’s geographical proximity to Japan and prior colonization by it for
half a century mean that Japanese culture has had a profound influence
on Taiwanese food. Accordingly, sushi is very popular in Taiwan. Many
conveyor-belt style sushi restaurant chains operate in Taiwan, including
the restaurant chains SUSHIRO, HAMA and KURA SUSHI. Currently,
there are almost 300 branches of a combined eight major conveyor-belt
sushi restaurants in Taiwan. In 2019 alone, the 27 KURA SUSHI
branches in Taiwan sold more than 46 million plates of sushi (https:
//www.foodnext.net/news/industry/paper/5616517067). Fish substi­
tution and mislabeling are practiced in the sushi industry, though rates
vary across regions; 3.4% in Italy (Armani et al., 2017), 10% in the UK
(Vandamme et al., 2016), 17.8% in Hong Kong (But, Wu, & Shaw,
2019), 23% in Metropolitan Vancouver, Canada (Hu et al., 2018), 26%
in Brazil (Staffen et al., 2017), and 47% in Los Angeles, USA (Willette
et al., 2017). Previous DNA barcoding studies have revealed that seafood
fraud is also a significant issue in the Taiwanese market (C.-H. Chang,
Lin, Ren, Lin, & Shao, 2016; P.-Y. Chen et al., 2020), as supported by
government inspections (https://www.mohw.gov.tw/cp-2623-196
14-1.html, https://foodtracer.taipei.gov.tw/Front/News/Detail?
id=1801).
A ~650-base pair (bp) region of the mitochondrial gene encoding for
cytochrome c subunit I (COI) has been employed as a taxonomic DNA
“barcode” (Ward, Hanner, & Hebert, 2009) and serves as a core element
for bioidentification of many Metazoan species. DNA-based species
authentication displays several advantages over traditional morpho­
logical approaches. DNA can be obtained from a tiny piece of tissue, is
more resistant to degradation and food processing, and DNA characters
are constant throughout a species’ lifetime. Moreover, current advances
in molecular biology technologies and computer software make it quite
easy to sequence DNA segments and compare them (C. H. Chang et al.,
2017). Moreover, an internationally-coordinated project to establish
standardized DNA barcodes for authoritatively-verified fish voucher
specimens, called the Fish Barcode of Life Initiative (FISH-BOL), has
been undertaken to provide a reference library for all fishes (Ward et al.,
2009). Today, DNA barcoding is frequently applied to explore mis­
labeling of fish products (Barendse et al., 2019; But et al., 2019; Liou
et al., 2020; Pardo & Jiménez, 2020; Shehata et al., 2018; Xiong et al.,
2019; Xiong et al., 2020).
Seafood fraud is a notable issue in Taiwan and sushi is widely
consumed there, but a mislabeling survey on sushi had not been con­
ducted previously in Taiwan. Moreover, mislabeling studies on sushi in
other countries did not focus on a specific type of restaurant. Therefore,
the goal of this study was to assess levels of fish substitution at conveyor-
belt sushi restaurants in Taiwan by means of DNA barcoding.
2
Food Control 130 (2021) 108264
2. Materials and methods
2.1. Sample collection
Between June to August 2020, we purchased a total of 122 tissue
samples from one restaurant of each of 11 different conveyor-belt sushi
chains located in four different cities of Taiwan: Taipei, Taoyuan, Tai­
chung, and Kaohsiung. Only fish-based sushi items were sampled.
Generally, an item of sushi only contains one kind of seafood, but some
may have both fish flesh and roe, in which cases both ingredients were
sampled. Thus, most samples represent individual sushi items (Table 1),
but four tissue samples, CB10/CB11 and TF07/TF08, were derived from
two sushi items. Information on the fish species of sushi items was
ascertained mainly from the menus, which were typically in Chinese. If
information on sushi items was available in English and/or Japanese
Kanji characters, such as “穴子 (representing conger eel), that was also
recorded. None of the restaurants provided scientific names of the fish
species they served.
Except for the sushi samples from Restaurant CB that were brought
back to the laboratory for sampling, all other samples were handled
immediately upon being served at the restaurants. The sushi samples
were first photographed using a smart phone and then tissue samples
were removed using autoclaved dissection tools and preserved in 99.5%
ethanol. The tissue samples were then brought back to the laboratory for
storage at − 20 ◦ C until DNA extraction. Images of the sushi samples are
presented in Supplementary Information 1.
2.2. DNA barcoding
DNA was extracted from each of the 122 tissue samples using DNA
extraction Kit S (Cat No./ID: GS100, Geneaid). PCR amplifications of the
partial mitochondrial COI gene (approximately 650 bp) were performed
in a mixture containing 5 ng template DNA, 12.5 μL of 2x Taq PCR
MasterMix (GN-PCR201-01, Genomix), and 12.5 μmol of each forward
and reverse primer—forward: FishF1+2 (5′ -TCR ACY AAY CAY AAA
GAY ATY GGC AC-3′ ); reverse: FishR1 (5′ - TAG ACT TCT GGG TGG CCA
AAG AAT CA-3′ ) or FishR2 (5′ -ACT TCA GGG TGA CCG AAG AAT CAG
AA-3′ ) (C.-H. Chang et al., 2016)—made up to a final volume of 25 μL
using distilled water. Thermal cycling began with one cycle at 95 ◦ C for
4 min, followed by 35 cycles of denaturation at 95 ◦ C for 30 s, 45–55 ◦ C
for 30 s, and 72 ◦ C for 30 s and, finally, a single extension step at 72 ◦ C
for 7 min. PCR products were purified using a PCR DNA Fragment
Extraction Kit (Geneaid, Taipei, Taiwan). Sequencing was performed by
Mission Biotech Inc., Taipei, Taiwan with the forward primer used for
PCR. The primer sequences linked to the amplified COI barcode
sequence of each sample were trimmed using 4 Peaks software. COI
sequences are given in Supplementary Information 2, but they have not
been submitted to GenBank since they are not derived from voucher
samples.
2.3. Data analysis
Edited COI sequences were compared to data in the public record
database of the Barcode of Life Database (BOLD) (http://www.boldsys
tems.org/) to establish species identification. Only matches with simi­
larity above 99% and with unambiguous species-level scientific names
were considered positive fish identifications. If more than one fish spe­
cies was shown as a positive match, all of those fishes were considered
potential candidates (Table 1). It has been demonstrated previously that
Neighbor-joining (NJ) analyses based on Kimura two-parameter (K2P)
distances using COI sequences cannot discriminate tunas (Thunnus spp.),
but mitochondrial control region sequences can (Mitchell & Hellberg,
2016; Pedrosa-Gerasmio, Babaran, & Santos, 2012; Viñas & Tudela,
2009). Fortunately, tuna species identification can still be achieved
using COI sequences via nucleotide composition at some diagnostic sites.
When BOLD identified one of our tissue samples as coming from tuna,
 C.-H. Chang et al.
Table 1
Sampled restaurants and details of 122 sushi samples.
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

2020.6.21 CA Kaohsiung CA01 鮭魚 raw Salmon Salmo salar + Oncorhynchus 100 Category I NO
City mykiss
CA02 鮪魚 raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
CA03 日本真鯛 raw Red sea bream Pagrus major + Pagrus major 100 NO YES
Oreochromis 100
niloticus
CA04 長鰭鮪 raw Longfin tuna Thunnus alalunga + Thunnus alalungae 100 NO YES
Thunnus obesus 100
Thunnus orientalis 99.82
Thunnus thynnus 99.82
Thunnus maccoyii 99.82
CA05 鰈魚邊緣 raw Flounder, Pleuronectes yokohamae + Atheresthes 100 NO NO
halibut, sole or Paralichthys stomias
olivaceus
CA06 日本產竹筴 raw Japanese jack Trachurus japonicus + Trachurus 100 NO YES
魚 mackerel japonicus
Trachurus declivis 100
Trachurus 99.83
novaezelandiae
Trachurus 99.32
mediterraneus
CA07 星鰻 simmered Conger Conger myriaster + Conger myriaster 100 NO YES
CA08 kabayaki Eel Anguilla japonica Anguilla japonica 100 NO YES
3

極上鰻魚 +
CA09 鯡魚卵 marinated Herring roe Clupea pallasii + Clupea pallasii 100 NO YES
CA10 飛魚卵 marinated Flying fish roe Exocoetidae + Prognichthys sealei 99.17 NO YES
Cheilopogon 99
furcatus
CA11 特選鮪魚中 raw Tuna Thunnus spp. + Thunnus thynnuse 100 NO YES
腹
Thunnus orientalis 100
CA12 鹽醋漬鯖魚 marinated Mackerel Scomber spp. + Scomber japonicus 100 NO YES
Scomber colias 99.01

2020.6.23 CB Kaohsiung CB01 鮭魚 Salmon raw Salmon Salmo salar + Salmo salar 100 NO YES
City
CB02 鮪魚 Tuna raw Tuna Thunnus spp. + Thunnus 99.83 NO YES
albacarese
CB03 旗魚 Marlin raw Marlin, billfish, Istiophoriformes + Makaira nigricans 100 NO YES
swordfish
Istiompax indica 100
CB04 鯛魚 Tilapia raw Snapper, sea Pagrus major + Oreochromis 100 Category I NO
bream aureus

Food Control 130 (2021) 108264

Oreochromis 100
niloticus
CB05 竹筴魚 Yellow stripe raw Japanese jack Trachurus japonicus + Selaroides 100 Category I NO
scad mackerel leptolepis
Caranx 99.82
sexfasciatus
CB06 泰式金目鱸 raw Sea bass Lates calcarifer + Lates uwisaraf 100 Category NO
II
Lates calcarifer 100
(continued on next page)
 C.-H. Chang et al.
Table 1 (continued )
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

CB07 醋烤鯖魚 Grilled pickled marinated Mackerel Scomber spp. + Scomber scombrus 99.82 NO YES
mackerel
CB08 鰻魚 Eel kabayaki Eel Anguilla japonica + Anguilla anguilla 100 NO NO
CB09 蒲燒星鰻 Kabayaki kabayaki Conger Conger myriaster + Conger myriaster 100 NO YES
conger eel
CB10a 黃金鲱魚 Herring with marinated Herring Clupae pallasii + Mallotus villosus 100 Category I NO
(roe part) capelin roe
CB11a 黃金鲱魚 Herring with marinated Herring Clupae pallasii + Mallotus villosus 100 Category I NO
(fish flesh capelin roe
part)

2020.7.04 CC Taoyuan CC01 青魽 raw Japanese Seriola quinqueradiata + Seriola 99.83 NO YES
City amberjack quinqueradiata
CC02 鮭魚 raw Salmon Salmo salar + Salmo salar 100 NO YES
CC03 鮪魚 raw Tuna Thunnus spp. + Thunnus obesuse 100 NO YES
Thunnus albacares 99.67
Thunnus atlanticus 99.6
CC04 鮮鰹魚 raw Skipjack tuna Katsuwonus pelamis + Sarda orientalis 100 Category I NO
CC05 鰈魚鰭邊 raw Flounder, Pleuronectes yokohamae + Reinhardtius 100 NO NO
halibut, sole or Paralichthys hippoglossoides
olivaceus
CC06 日本產真鯛 raw Red sea bream Pagrus major + Pagrus major 100 NO YES
Oreochromis 99.66
niloticus
CC07 raw Tuna Thunnus spp. Thunnus thynnuse 100 NO YES
4

鮪魚中腹 +
Thunnus orientalis 99.84
CC08 魩仔魚軍艦 raw Whitebait Clupeiformes + Engraulis japonicus 99.83 NO YES
CC09 烤鱒魚腹 grilled Trout Oncorhynchus mykiss + Salmo salar 100 Category I NO
CC10 青花魚 marinated Mackerel Scomber spp. + Scomber scombrus 99.83 NO YES
CC11 星鰻 simmered Conger Conger myriaster + Conger myriaster 100 NO YES
CC12 青魽腹脂 raw Japanese Seriola quinqueradiata + Seriola 99.83 NO YES
amberjack quinqueradiata
CC13 柳葉魚卵軍 marinated Willow leaf fish Spirinchus lanceolatus + Mallotus villosus 100 Category I NO
艦 (shishamo)
CC14 鰻魚卷 kabayaki Eel Anguilla japonica + Anguilla japonica 100 NO YES
CC15 鱈魚美乃滋 marinated Gadoid fish Gadus chalcogrammus + Gadus 100 NO YES
軍艦 chalcogrammus

2020.7.7 TA Taichung TA01 鮭魚肚 Salmon belly raw Salmon Salmo salar + Salmo salar 100 NO YES
City
TA02 鰈魚緣側 Flounder fin raw Flounder, Pleuronectes yokohamae + Reinhardtius 100 NO NO
halibut, sole or Paralichthys hippoglossoides
olivaceus
TA03 長鰭鮪魚 Albacore raw Longfin tuna Thunnus alalunga + Thunnus alalungae 100 NO YES

Food Control 130 (2021) 108264

Thunnus obesus 99.83
Thunnus orientalis 99.66
Thunnus thynnus 99.66
Thunnus maccoyii 99.66
TA04 白皮旗魚 raw Black marlin Istiompax indica + Istiompax indica 100 NO YES
Istiophorus 99.82
platypterus
TA05 熟成鮪魚 Tuna raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
(continued on next page)
 C.-H. Chang et al.
Table 1 (continued )
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

TA06 秋刀魚 Saury raw Pacific saury Cololabis saira + Cololabis saira 100 NO YES
TA07 熟成黑鮪魚 Bluefin tuna raw Bluefin tuna Thunnus orientalis + Thunnus thynnuse 100 NO NO
中肚 chutoro
Thunnus orientalis 100
TA08 星鰻 Grilled conger simmered Conger Conger myriaster + Conger myriaster 100 NO YES
eel
TA09 柳葉魚卵軍 Seasoned marinated Willow leaf fish Spirinchus lanceolatus + Mallotus villosus 100 Category I NO
艦 capelin (shishamo)
TA10 紅魽 raw Great amberjack Seriola dumerili + Seriola dumerili 100 NO YES
Lichia amia 99.33
TA11 金華鯖魚 Seared fatty marinated Mackerel Scomber spp. + Scomber japonicus 100 NO YES
mackerel

2020.7.8 TB Taichung TB01 鰤魚 Yellowtail raw Amberjack Seriola quinqueradiata + Seriola 100 NO YES
City quinqueradiata
TB02 鮭魚 Salmon raw Salmon Salmo salar + Salmo salar 100 NO YES
TB03 鮪魚 Tuna raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
TB04 蒲燒鰻魚 Grilled eel kabayaki Eel Anguilla japonica + Anguilla anguilla 100 NO NO

2020.7.9 TC Taichung TC01 蒲燒鰻魚 Grilled eel fish kabayaki Eel Anguilla japonica + Anguilla 100 NO NO
City marmorata
TC02 蒲燒秋刀魚 Grilled saury kabayaki Pacific saury Cololabis saira + Cololabis saira 100 NO YES
TC03 鮭魚肚 Fatty salmon raw Salmon Salmo salar + Salmo salar 100 NO YES
TC04 Tuna raw Tuna Thunnus spp. Thunnus 100 NO YES
5

鮪魚 +
albacarese
TC05 日本青魽 Amberjack fish raw Japanese Seriola quinqueradiata + Seriola 99.83 NO YES
amberjack quinqueradiata
TC06 軍曹魚 Ceder fish raw Cobia Rachycentron canadum + Rachycentron 100 NO YES
canadum
TC07 星鰻 Eel simmered Conger Conger myriaster + Conger myriaster 99.83 NO YES
TC08 雙色海鱺 Two types of raw Cobia Rachycentron canadum + Rachycentron 100 NO YES
cobla canadum
TC09 山葵比目魚 Flounder fish raw Flounder, Pleuronectes yokohamae + Reinhardtius 99.83 NO NO
with wasabi halibut, sole or Paralichthys hippoglossoides
olivaceus
TC10 日本真鯛 Red sea bream raw Red sea bream Pagrus major + Pagrus major 100 NO YES
fish
Oreochromis 99.66
niloticus
TC11 黃金白魚 Golden raw Noodlefish Salangichthys microdon + Neosalanx 100 NO NO
whitebait brevirostris
Neosalanx 100
tangkahkeii
TC12 Fatty tuna raw Tuna Thunnus spp. Thunnus 100 NO YES

Food Control 130 (2021) 108264

鮪魚中腹 +
albacarese

2020.7.16 CD Taipei City CD01 鮭魚 raw Salmon Salmo salar + Salmo salar 100 NO YES
CD02 鮪魚 raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
CD03 日本鰤魚 raw Japanese Seriola quinqueradiata + Seriola 100 NO YES
amberjack quinqueradiata
CD04 日本真鯛 raw Red sea bream Pagrus major + Pagrus major 100 NO YES
(continued on next page)
 C.-H. Chang et al.
Table 1 (continued )
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

Oreochromis 100
niloticus
CD05 海鱺 flamed Cobia Rachycentron canadum + Rachycentron 100 NO YES
canadum
CD06 蒲燒鰻魚 kabayaki Eel Anguilla japonica + Anguilla anguilla 100 NO NO
CD07 炙燒鰈魚緣 flamed Flounder, Pleuronectes yokohamae + Reinhardtius 100 NO NO
側 halibut, sole or Paralichthys hippoglossoides
olivaceus
CD08 日本鰤魚肚 raw Japanese Seriola quinqueradiata + Seriola 99.67 NO YES
amberjack quinqueradiata
CD09 星鰻 simmered Conger Conger myriaster + Conger myriaster 100 NO YES
CD10 竹莢魚 raw Japanese jack Trachurus japonicus + Decapterus akaadsi 100 Category I NO
mackerel
Decapterus 100
maruadsi
Decapterus 100
macarellus
CD11 青花魚 marinated Mackerel Scomber spp. + Scomber japonicus 100 NO YES
CD12 黑鮪 raw Bluefin tuna Thunnus orientalis + Thunnus thynnuse 100 NO NO
Thunnus orientalis 100
2020.7.27 TD Taipei City TD01 鮭魚 raw Salmon Salmo salar + Salmo salar 100 NO YES
TD02 海鱺 raw Cobia Rachycentron canadum + Rachycentron 100 NO YES
canadum
TD03 鮪魚 raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
6

TD04 紅甘 raw Great amberjack Seriola dumerili + Seriola dumerili 100 NO YES
Lichia amia 99.33
TD05 漬鯖魚 marinated Mackerel Scomber spp. + Scomber scombrus 100 NO YES
TD06 蒲燒鰻 kabayaki Eel Anguilla japonica + Anguilla japonica 100 NO YES
TD07 比目魚鰭邊 raw Flounder, Pleuronectes yokohamae + Reinhardtius 100 NO NO
肉 halibut, sole or Paralichthys hippoglossoides
olivaceus
TD08 明太子 marinated Pollock roe Gadus chalcogrammus + Mallotus villosus 100 Category I NO
(Mentaiko)

2020.07.29 TE Taipei City TE01 鮭魚 Salmon raw Salmon Salmo salar + Salmo salar 100 NO YES
TE02 穴子 Conger Eel simmered Conger Conger myriaster + Conger myriaster 100 NO YES
TE03 鮪魚 Tuna raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
TE04 漬鯖魚 Pickled club marinated Mackerel Scomber spp. + Scomber scombrus 100 NO YES
mackerel
TE05 青甘 raw Japanese Seriola quinqueradiata + Seriola 100 NO YES
amberjack quinqueradiata
TE06 七星鱸魚 Sea Bass raw Japanese sea Lateolabrax japonicus + Lateolabrax 100 NO YES
bass japonicus

Food Control 130 (2021) 108264

TE07 比目魚鰭邊 Flounder flamed Flounder, Pleuronectes yokohamae + Reinhardtius 100 NO NO
肉 Dorsal Fin Ray halibut, sole or Paralichthys hippoglossoides
olivaceus
TE08 黑鮪-赤身 Bluefin Tuna raw Bluefin tuna Thunnus orientalis + Thunnus orientalise 100 NO YES
Thunnus thynnus 100
Thunnus maccoyii 100
Thunnus alalunga 99.82
Thunnus obesus 99.82
(continued on next page)
 C.-H. Chang et al.
Table 1 (continued )
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

TE09 紅甘 Rudderfish raw Great amberjack Seriola dumerili + Seriola dumerili 100 NO YES
great
amberjack
Lichia amia 99.34
TE10 鰻魚 Eel kabayaki Eel Anguilla japonica + Anguilla japonica 100 NO YES
TE11 旗魚 Marlin raw Marlin, billfish, Istiophoriformes + Makaira nigricans 100 NO YES
swordfish
Istiompax indica 100
TE12 海鱺 Cobia raw Cobia Rachycentron canadum + Rachycentron 100 NO YES
canadum
TE13 鰹魚 Skipjack tuna raw Skipjack tuna Katsuwonus pelamis + Euthynnus affinis 100 Category I NO
Euthynnus lineatus 99.6
TE14 竹莢魚 Jack mackerel raw Japanese jack Trachurus japonicus + Decapterus 100 Category I NO
mackerel maruadsi
Decapterus 100
kurroides
2020.07.30 CE Taipei City CE01 大關竹莢魚 raw Japanese jack Trachurus japonicus + Trachinotus ovatus 100 Category I NO
mackerel
Trachinotus anak 100
CE02 黃雞魚 raw Chicken grunt Parapristipoma + Trachinotus ovatus 100 Category I NO
trilineatum
Trachinotus anak 100
CE03 鰹魚 raw Skipjack tuna Katsuwonus pelamis + Katsuwonus 100 NO YES
pelamis
CE04 marinated Mackerel Scomber spp. Scomber scombrus 100 NO YES
7

鯖魚 +
CE05 鰈魚緣側 flamed Flounder, Pleuronectes yokohamae + Reinhardtius 99.83 NO NO
halibut, sole or Paralichthys olivaceus hippoglossoides
CE06 鮭魚 raw Salmon Salmo salar + Salmo salar 99.83 NO YES
CE07 飛魚卵 raw Flying fish roe Exocoetidae + Mallotus villosus 100 Category I NO
CE08 黑鮪魚中腹 raw Bluefin tuna Thunnus orientalis + Thunnus thynnuse 100 NO NO
Thunnus orientalis 100
CE09 真鯛 raw Red sea bream Pagrus major + Pagrus major 100 NO YES
Oreochromis 99.82
niloticus
CE10 鰻魚 kabayaki Eel Anguilla japonica + Anguilla japonica 100 NO YES
CE11 鮪魚 raw Tuna Thunnus spp. + Thunnus 100 NO YES
albacarese
CE12 星鰻 simmered Conger Conger myriaster + Conger myriaster 100 NO YES

2020.08.06 TF Taichung TF01 鹽烤秋刀魚 grilled Pacific saury Cololabis saira - - - - -

City
TF02 蒲燒鰻魚軍 kabayaki Eel Anguilla japonica + Anguilla japonica 100 NO YES
艦
TF03 蝦味魚卵軍 marinated Fish roe Fish + Mallotus villosus 100 NO YES

Food Control 130 (2021) 108264

艦
TF04 旗魚 raw Marlin, billfish, Istiophoriformes + Makaira nigricans 100 NO YES
swordfish
Istiompax indica 100
TF05 鮭魚 raw Salmon Salmo salar + Salmo salar 100 NO YES
TF06 鮪魚 raw Tuna Thunnus spp. + Thunnus obesuse 100 NO YES
Thunnus albacares 99.83
Thunnus atlanticus 99.8
TF07b marinated Herring Clupae pallasii + Mallotus villosus 100 Category I NO
(continued on next page)
 C.-H. Chang et al.
Table 1 (continued )
Date Restaurant Sampling Sample Chinese English label Product English Expected speciesd PCR BOLD (identified Similarity Mislabeled Expected
code locality code label type translation of amplification as) %
Chinese labelc

黃金鲱魚
(roe part)
b
TF08 黃金鲱魚 marinated Herring Clupae pallasii + Mallotus villosus 100 Category I NO
(fish flesh
part)
TF09 鯛魚 raw Snapper, sea Pagrus major + Oreochromis 100 Category I NO
bream aureus
Oreochromis 100
niloticus
Oreochromis 100
mossambicus
TF10 燒烤多利魚 grilled Dory Zeidae + Pangasianodon 100 Category I NO
hypophthalmus
8

TF11 炙燒金目鱸 flamed Sea bass Lates calcarifer + Lates calcariferf 100 NO YES
a,b
Denotes separate samples from the same sushi item, respectively.
c
English common names of the labeled Chinese names were translated by using the Fish Database of Taiwan (https://fishdb.sinica.edu.tw/), A Guide Book of Common Economic Aquatic Animals and Plants in Taiwan
(Shao et al., 2015), and the Sushi Gourmet Handbook (Fukuchi, 2014).

d
The expected species of each sushi item was assessed according to its label and the Sushi Gourmet Handbook (Fukuchi, 2014).

e
Species authenticated based on the character-based diagnostic methods (Lowenstein et al., 2009; Puncher et al., 2015).

f
Species authenticated based on the Neighbor-joining (NJ) phylogenetic analysis (Fig. 1).

Food Control 130 (2021) 108264

C.-H. Chang et al. Food Control 130 (2021) 108264

the character-based barcoding methods established by Lowenstein, Table 2

Amato, and Kolokotronis (2009) and Puncher et al. (2015) were Summary of Chinese labels, English translations, and expected species from
employed to further authenticate tuna species identity. Table 1.
When BOLD identified tissue samples as being derived from Lates Chinese label English translation of Expected speciesb
spp. (sea bass, in Chinese: 金目鱸), a NJ analysis was conducted using Chinese labela
the two newly acquired COI sequences (CB06 and TF11, this study) 鮭魚 Salmon Salmo salar
combined with another 14 COI sequences generated by Ward, Holmes, 鱒魚 Trout Oncorhynchus mykiss
and Yearsley (2008) for L. uwisara (GenBank EF609378 to EF609382), 鮪魚 Tuna Thunnus spp.
Bluefin tuna Thunnus orientalis
L. calcarifer (GenBank DQ108012, DQ108013, DQ108026, and 黑鮪
長鰭鮪 Longfin tuna Thunnus alalunga
DQ108027), and L. niloticus (GenBank DQ108015 to DQ108019). These 鰹魚 Skipjack tuna Katsuwonus pelamis
16 COI sequences were aligned using MACSE v2 software (Ranwez, 旗魚 Marlin, billfish, swordfish Istiophoriformes
Douzery, Cambon, Chantret, & Delsuc, 2018). A NJ analysis based on 白皮旗魚 Black marlin Istiompax indica
K2P distances was used to build a distance tree with 1,000,000 bootstrap 真鯛 Red sea bream Pagrus major
Snapper, sea bream Pagrus major
replicates in MEGA X (S. Kumar, Stecher, Li, Knyaz, & Tamura, 2018).
鯛魚
星鰻、穴子 Conger Conger myriaster
鰻魚 Eel Anguilla japonica
2.4. Comparison of analytical results and product labels 鰈魚、比目 Flounder, halibut, sole Pleuronectes yokohamae or
魚 Paralichthys olivaceus
Japanese jack mackerel Trachurus japonicus
The results of our molecular identification of each sample were then
竹筴魚
秋刀魚 Pacific saury Cololabis saira
compared to the Chinese common names labeled on the menus from the 鯡魚 Herring Clupea pallasii
sampling restaurants. Since a Taiwan government-approved standard 飛魚 Flying fish Exocoetidae
for common names of fishes does not exist, the English names of the 鯖、青花魚 Mackerel Scomber spp.
labeled Chinese common names were consulted by using the fish data­ 金目鱸 Sea bass Lates calcarifer
魩仔魚 Whitebait Clupeiformes
base of Taiwan (https://fishdb.sinica.edu.tw/), A Guide Book of Com­ 柳葉魚 Willow leaf fish (shishamo) Spirinchus lanceolatus
mon Economic Aquatic Animals and Plants in Taiwan (Shao et al., 鱈魚 Gadoid fish Gadus chalcogrammus
2015), and the Sushi Gourmet Handbook (Fukuchi, 2014). Moreover, 軍曹魚、海 Cobia Rachycentron canadum
although a common name can often be applied to more than one fish 鱺
Amberjack Seriola quinqueradiata
species, a sushi item generally refers to a specific fish species (Fukuchi,
鰤魚
紅魽、紅甘 Great amberjack Seriola dumerili
2014). For example, traditionally, kabayaki-style eel (filleted eel that is 青魽、日本 Japanese amberjack Seriola quinqueradiata
steamed and then grilled with a sweet sauce) (in Chinese: 蒲燒鰻) is 鰤魚
made of Japanese eel (Anguilla japonica) rather than other Anguilla 七星鱸魚 Japanese sea bass Lateolabrax japonicus
species. Thus, a kabayaki-style eel sample was “correctly labeled” if it 黃雞魚 Chicken grunt Parapristipoma trilineatum
Noodlefish Salangichthys microdon
did indeed contain eel, but if it did not specifically contain A. japonica it
黃金白魚
多利魚 Dory Zeidae
was considered not to meet “customer expectations”. Therefore, the 明太子 Pollock roe (Mentaiko) Gadus chalcogrammus
expected fish species of each sushi item according to its label and the 魚 Fish Fish
Sushi Gourmet Handbook (Fukuchi, 2014) was also compared to the a
English common names of the labeled Chinese names were translated by
barcode-authenticated species. A summary of Chinese labels, English using the Fish Database of Taiwan (https://fishdb.sinica.edu.tw/), A Guide Book
translations, and the expected species of all collected sushi items is of Common Economic Aquatic Animals and Plants in Taiwan (Shao et al., 2015),
presented in Table 2. A sample was deemed mislabeled when the and the Sushi Gourmet Handbook (Fukuchi, 2014).
barcode-authenticated species name did not match the name on the
b
menu. The mislabeling cases were further classified into two categories The expected species of each sushi item was assessed according to its label
based on (Günther, Raupach, & Knebelsberger, 2017). Category I and the Sushi Gourmet Handbook (Fukuchi, 2014).
comprised instances where the labeled common name and
barcode-authenticated species were in different genera. Category II re­
flected instances where both the menu-declared and length of 618 bp and a minimum length of 471 bp. All 121 samples could
barcode-authenticated names were in the same genus. be identified to species level using the BOLD identification tool and
additional methodologies, and 84 barcoding segments matched a single
3. Results and discussion species under our 99% similarity threshold.
DNA barcoding efficiency depends on two factors, i.e., natural lim­
3.1. Data analysis itations and human error. First, the discriminatory power of DNA bar­
coding relies on the gap between COI interspecific diversity and
DNA is somewhat degenerated by food processing, which limits intraspecific diversity, which is termed the “barcoding gap”. Conse­
conventional full-length DNA barcoding protocols that target ~650-bp quently, if this gap is not sufficiently distinct due to hybridization or
region of the COI gene. Therefore, COI mini-barcodes, ranging from 100 incomplete lineage sorting, there can be some ambiguity in species as­
to 400 bp, have frequently been employed as an alternative method for signments. Second, having a complete and accurate reference database
molecular authentication of processed foods (Armani et al., 2017; is critical to attaining accurate DNA barcoding results. If reference se­
Günther et al., 2017; Sultan et al., 2018; Vandamme et al., 2016; Xing, quences have incorrect species names, whether due to specimen
Hu, Han, Deng, & Chen, 2020; Xiong et al., 2019). Although 46 of our misidentification or typographical error, so that a reference barcode
tissue samples had undergone some type of processing, such as by sequence will not match the correct scientific name, then the database
marinating, flaming, or grilling, we obtained 121 barcoding COI se­ will present incorrect authentications (Fernandes, Amaral, & Mafra,
quences from the 122 tissue samples in amplifying full-length DNA 2021). In this study, both natural limitations and human/database error
barcodes, with only one of the processed samples (TF01) failing to have hampered our DNA barcoding efforts.
amplify. A high success rate in obtaining interpretable sequences from Both Thunnus spp. (tuna) and Trachurus spp. (jack mackerel) suffer
sushi samples has also been reported in other studies (Armani et al., from the natural limitations of DNA barcoding. Fortunately, character-
2017; But et al., 2019; Hu et al., 2018; Staffen et al., 2017; Vandamme based diagnostic methods have been proposed to offset some of those
et al., 2016; Willette et al., 2017). After trimming, the average length of limitations. By employing the methods proposed by Lowenstein et al.
these segments was 593.2 ± 21.5 bp (mean ± S.D), with a maximum (2009) and Puncher et al. (2015), we successfully identified all tuna

9
C.-H. Chang et al.
samples to species level. However, the diagnostic method created by
Neira, Perry, Burridge, Lyle, and Keane (2015) can only distinguish
T. declivis from T. novaezelandiae and it relies on cytochrome b rather
than COI sequences. The BOLD NJ tree-based identification of sample
CA06 demonstrates that it clusters with other T. japonicus sequences
rather than T. declivis, T. novaezelandiae, or T. mediterraneus, so we are
confident that it was correctly labeled on the menu as T. japonicus.
Our DNA barcoding results for sushi samples of red sea bream, marlin
(Istiophoriformes), and great amberjack probably suffer from database
errors. Although all four marlin-labeled samples (CB03, TA04, TE11,
and TF04) were barcode-authenticated as either blue marlin (Makaira
nigricans) or black marlin (Istiompax indica), the barcode sequences for
samples CB03, TE11, and TF04 matched multiple blue marlin reference
sequences but only one black marlin sequence. Therefore, it is very
likely that this black marlin reference sequence (BOLD Sequence ID:
FOA896-04.COI-5P) actually originated from blue marlin. Likewise, for
sample TA04, multiple reference sequences from black marlin matched
the query sequence, but there was also one Indo-Pacific sailfish (Istio­
phorus platypterus; GenBank accession: FJ265861) sequence with >99%
similarity, which might also be an error. Consequently, we suggest that
samples CB03, TE11, and TF04 are from blue marlin and sample TA04 is
derived from black marlin.
Mislabeled reference sequences in BOLD may explain why we could
not attain species-specific identities for samples labeled as great
amberjack (TA10, TD04, and TE09). Moreover, it is highly likely that the
BOLD reference sequence (GenBank accession: MT525054) marked as
Oreochromis niloticus is actually from red sea bream, explaining why
barcoding results for red sea bream-labeled samples matched multiple
red sea bream references and this sole O. niloticus sequence. A barcoding
reference sequence for Lichia amia (GenBank accession: JQ623944) in
the BOLD database is also likely incorrectly labeled as great amberjack.
However, certain ambiguities in our barcoding results cannot be so
easily assigned to natural limitation and/or human errors. For example,
many of our jack mackerel-labeled samples (including TE14, CE01,
CB05, and CD10) were assigned to more than one candidate carangidid
fish. It is unclear if these candidate species are hybrids, which are
difficult to identify correctly based on morphology, or if they have
complex taxonomic histories, but our findings clearly support that these
“jack mackerel” sushi items are mislabeled. Thus, although BOLD rep­
resents a useful database and tool for molecular authentication, spurious
reference sequences erode its utility in DNA barcoding exercises (Kappel
& Schröder, 2020). As suggested by Shehata et al. (2018) and Mitchell
et al. (2019), we recommend that the Taiwanese government establishes
its own reliable and complete DNA barcoding database for future au­
thentications of seafood resources.
In view of the extreme similarity among tuna barcoding segments,
the efficacy of DNA barcoding is greatly limited in terms of dis­
tinguishing tuna at species level. All 19 tissue samples labeled as tuna (in
Chinese: 鮪魚) were confirmed as being Thunnus spp. based on BOLD
analysis. The precise species corresponding to these 19 samples was
pinpointed using the character-based methods proposed by Lowenstein
et al. (2009) and Puncher et al. (2015), which revealed five different
tuna species in our sample set: longfin tuna (T. alalunga), bigeye tuna
(T. obesus), yellowfin tuna (T. albacares), Atlantic bluefin tuna
(T. thynnus), and Pacific bluefin tuna (T. orientalis). Previous barcoding
studies on sushi have revealed that tuna is frequently substituted with
escolar (Lepidocybium flavobrunneum), salmon (Salmo salar), banded
rudderfish (Seriola zonata), great amberjack (Seriola dumerili), skipjack
tuna (Katsuwonus pelamis), little tunny (Euthynnus alletteratus), various
shark species, or that relatively cheaper tuna species are sold as bluefin
tuna (Delpiani et al., 2020; Hu et al., 2018; Minoudi et al., 2020; Pardo &
Jiménez, 2020; Staffen et al., 2017; Vandamme et al., 2016; Willette
et al., 2017; Xing et al., 2020). Our DNA barcoding analysis has revealed
that all of the tuna-labeled items we sampled truly contain flesh from
Thunnus fishes and, furthermore, all of the bluefin tuna-labeled items
also indeed came from two of three bluefin tuna species. However, we
10
Food Control 130 (2021) 108264
mostly sampled Atlantic bluefin tuna (T. thynnus), meaning that the
sushi used in the restaurants we assessed is not sourced locally. Both the
Taiwanese and Japanese governments promote bluefin tuna activities,
such as the Pintung Bluefin Tuna Cultural Festival in Taiwan and the
Tuna Thanksgiving in Oma, Japan, so customers in sushi restaurants in
Taiwan likely expect to be served Pacific bluefin tuna (T. orientalis),
which can be caught in offshore areas of Taiwan and Japan. Importantly,
increased exposure to mercury and issues surrounding tuna conserva­
tion should prompt customers to limit their consumption of tuna in
Taiwanese restaurants (G. Kumar, 2018; Wakamatsu & Managi, 2019).
For the two sea bass sushi samples (CB06 and TF11), BOLD identi­
fication revealed that they are both Lates species. However, only sample
TF11 could be unambiguously linked to L. calcarifer, whereas sample
CB06 could be identified by BOLD as either L. uwisara or L. calcarifer
(Table 1). L. uwisara and L. calcarifer are very similar. L. uwisara was first
identified as a possible cryptic species within L. calcarifer based on COI
sequence analysis (Ward et al., 2008). It was formally published as a new
species upon detailed morphological assessment (Pethiyagoda & Gill,
2012). In this study, we adopted the phylogenetic method and sequences
used by Ward et al., 2008 to differentiate L. uwisara from L. calcarifer.
We considered a dataset of 16 Lates COI sequences with a total of 573
base pairs, which contained 123 variable sites and 121
parsimony-informative sites. The results of our NJ analysis showed that
our sample CB06 clustered among sequences corresponding to L. uwisara
and that our sample TF11 associated with L. calcarifer sequences
(bootstrap values ≥ 70; Fig. 1). Ambiguities in our raw barcoding results
for sample CB09 arose because reference barcode sequences for
L. uwisara submitted before its official recognition in 2012 are still
labeled as L. calcarifer. Our NJ analysis supports that sample CB09 is
derived from L. uwisara and that sample TF11 came from L. calcarifer.
3.2. Comparison of molecular results and labeling information
Overall, we report a mislabeling rate of 17.36% (21/121). Of 21
mislabeled samples, 20 were in Category I and only 1 was assigned to
Category II. Based on these cases of mislabeling, fish roe items, as well as
Japanese jack mackerel, are particularly prone to food substitution.
Indeed, six of the nine fish roe items we sampled were mislabeled. Those
six fish roe sushi items were either labeled as herring (in Chinese: 鯡魚;
samples: CB10, TF07), willow leaf fish (shishamo) (in Chinese: 柳葉魚;
CC13, TA09), flying fish (in Chinese: 飛魚; CE07), or pollock roe
(mentaiko) (in Chinese: 明太子; TD08), but in fact our barcoding showed
that the roe all came from capelin (Mallotus villosus). Only one (CA06) of
the five sushi items labeled as jack mackerel (in Chinese: 竹莢魚) was
molecularly identified as T. japonicus, with the other four samples
(TE14, CE01, CB05, and CD01) identified as other carangidid species.
Capelin roe is cheaper than roe from flying fish or herring, so financial
gain is very likely the motivation for substituting roe (Armani et al.,
2017; But et al., 2019). However, it is not clear to us why jack mackerel
might be mislabeled. We found that jack mackerel is substituted with
other carangidid fishes, such as species of Decapterus spp., Trachinotus
spp., or Selaroides spp.. Given that these substituting species are quite
similar to the labeled species, it is difficult to ascertain if these cases of
mislabeling are accidental or intentional.
Of the remaining 100 samples, in 84 the menu-described common
name matched the expected species identified from BOLD analysis.
However, we did identify 16 cases where customers may have been
served an “unexpected” fish species. For instance, sushi items labeled as
flatfish (in Chinese: 比目魚or 鰈魚) would be expected to comprise
marbled sole (Pseudopleuronectes yokohamae) or Japanese flounder
(Paralichthys olivaceus) (Table 2), yet our DNA analysis revealed them to
be arrowtooth flounder (Atheresthes stomias) or Greenland halibut
(Reinhardtius hippoglossoides). Similarly, we identified cases where
Anguilla species other than A. japonica were being used for kabayaki-
style eel, and where Atlantic bluefin tuna was being used instead of
Pacific bluefin tuna (in Chinese: 黑鮪).
C.-H. Chang et al.
Fig. 1. Neighbor-joining (NJ) tree of Lates species inferred from barcoding segments (573 bp) with 1,000,000 bootstrap replicates. Each terminal is labeled with the
sample code (this study) or the scientific name and its GenBank accession number. Bootstrap values are labeled on respective branches of the NJ tree.
Previous mislabeling studies have indicated that restaurants are
more vulnerable to food fraud than supermarkets and other retailers (Hu
et al., 2018; Pardo et al., 2016). However, the mislabeling rate (17.36%)
we estimated for conveyor-belt sushi restaurant chains in our study is
close to the gross mislabeling rate (18.9%) previously reported for
seafood in Taiwan (P.-Y. Chen et al., 2020). Moreover, this mislabeling
rate of 17.36% is moderate relative to those reported in other sushi
barcoding studies from different regions, such as 10% in the UK, 26.0%
in Hong Kong, and 23% in Vancouver (Armani et al., 2017; But et al.,
2019; Hu et al., 2018; Staffen et al., 2017; Vandamme et al., 2016;
Willette et al., 2017). We acknowledge that the mislabeling rate we
report herein may be an underestimate since some of the common names
in our study correspond to a whole taxonomic order or family of fish
species rather than a specific species and as some unexpected species
arose that though “correctly” labeled may not have matched customer
expectations. One such ambiguous Chinese common name is 鯛; apart
from denoting snapper or seabream, it is also an umbrella term for many
types of marine fishes such as the Lutjanidae (in Chinese: 笛鯛科), the
Priacanthidae (in Chinese: 大眼鯛科), and the Sparidae (in Chinese: 鯛
科), all of which commonly end with the symbol “鯛”. Consequently, all
of the species in these families could be labeled“鯛魚”. To improve both
domestic and foreign market access, tilapia in Taiwan has been labeled
as Taiwan-Snapper (in Chinese: 台灣鯛) since Taiwan joined the World
Trade Organization (WTO) in 2002. However, 台灣鯛 represents tilapia,
but the iconic species denoted by 鯛魚 for Taiwanese customers is red
sea bream (Pagrus major) (in Chinese: 真鯛), with this latter species also
traditionally being used as an auspicious food at weddings and during
New Year in Japan (Fujita, 1979). For our purposes, we assume that
sushi items labeled “鯛魚” should at least be snapper or sea bream but
not tilapia, and that customers would expect them to be red sea bream.
We did note that kabayaki-style eel items were made from three
different Anguilla species—Japanese eel, European eel (A. anguilla), and
giant mottled eel (A. marmorata)—even though, traditionally, only
Japanese eel is served kabayaki-style (Fukuchi, 2014). Shortages of
11
Food Control 130 (2021) 108264
Japanese, European, and American eel (A. rostrata) in markets and
increasing conservation concern mean that both European and Amer­
ican eels are appearing in sushi supply-chains in response to increasing
demand, and aquacultural production of giant mottled eel and shortfin
eel (A. bicolor) has been established (Huang, Liu, Chang, Yuan, & Miao,
2016). All five of these Anguilla species are being served in sushi res­
taurants (Armani et al., 2017; Vandamme et al., 2016).
Chinese is the primary language of Taiwan. Of the 11 sushi restau­
rant chains we sampled in, five also provide English labels, which
further exacerbated labeling complexity. We notice there are four main
issues in sushi labeling. First, the inconsistencies between Chinese and
English labels. As examples, sample CB04 had a Chinese label of “鯛”
(snapper/seabream and discussed in detail above) but an English label
of “tilapia”. Our barcoding analysis of these samples demonstrated that
the molecularly-authenticated species corresponds to the English labels
rather than their Chinese labels. These inconsistencies could represent a
loophole in labeling regulations. Second, we found cases of inconsistent
usage of Chinese common names. The Chinese characters “紅魽” and “紅
甘” both represent the same species, great amberjack (S. dumerili)
(Table 2), so even in a given restaurant, the same fish species could be
labeled with different Chinese characters. Moreover, in general, a
salmonid fish is termed “salmon (in Chinese: 鮭魚)” if it is anadromous
and “trout (in Chinese: 鱒魚)” if it is not. Confusingly, rainbow trout
(Oncorhynchus mykiss) has both resident and anadromous forms, so it
cannot be labeled as “salmon”, though instances of such mislabeling
have been reported from Chile, China, and Canada (Hu et al., 2018;
Prida et al., 2020; Xing et al., 2020). Here, we found that usage of 鮭魚
and 鱒魚 in Taiwan is not unified. A primary concern with labeling
rainbow trout as salmon is increased risk of zoonotic parasites (Cabello,
2007) since, unlike Atlantic salmon, rainbow trout is more likely to have
been raised in freshwater rather than seawater. Third, there were also
cases of ambiguous English labels on sushi menus. The English label for
sample TC06 is “ceder fish” and that of TC08 is “cobla”, but neither of
these names represent cobia (Rachycentron canadum). Fourth, the usage
C.-H. Chang et al.
of Japanese kanji might confuse consumers. Japanese kanji originates
from Chinese characters, and Chinese labeling of sushi items often
directly adopts the Japanese kanji symbols. For example, the Chinese
label associated with sample TE02, 穴子, is taken directly from the
Japanese kanji for Conger myriaster. However, when Japanese kanji and
Chinese characters do not correspond to the same fish species, labeling
can become confused. For example, in Japanese kanji,“鰤” solely rep­
resents Seriola quinqueradiata (Fukuchi, 2014), but, as a Chinese char­
acter, it can refer to Seriola spp. (in Chinese: 鰤屬) (Table 2). Here, we
assumed that Chinese characters have been used in the Chinese labels on
sushi menus. Therefore, species labeled as “鰤” represent all Seriola
fishes. Then, according to the Sushi Gourmet Handbook (Fukuchi,
2014), the expected species for 鰤-labeled items was deemed to be
S. quinqueradiata.
Countries such as Australia, Brazil, Italy, German, Spain, UK, and
USA all have official standards of common fish names (Armani et al.,
2017; Günther et al., 2017; Pardo & Jiménez, 2020; Staffen et al., 2017;
Vandamme et al., 2016; Willette et al., 2017). Even some commercial
fish terms have been clearly defined in some regions. For example, ac­
cording to Code of Federal Regulations Title 21 §102.57 of the US Food
& Drug Administration, the term “halibut” can only be used to describe
Hippoglossus hippoglossus and H. stenolepis. Similarly, only 22 fish species
can be deemed sardines based on CXS 94-1981 of the Codex Ali­
mentarius Commission (a joint FAO and WHO body) (fil­
e:///Users/chahao/Downloads/CXS_094e.pdf). Such standardization of
common fish names is crucial to avoid misleading consumers (FAO,
2020) and for prohibiting fish fraud (Reilly, 2018). The Food and Drug
Administration of Taiwan has officially declared that only fishes in the
Gadiformes can be labeled as cod (in Chinese: 鱈) (FDA食字第
1051302452號 https://www.tqf.org.tw/upload/content/FDA10513
02452.pdf), but it has yet to provide clear definitions for the different
Chinese characters for flatfish, such as 鰈魚 and 比目魚. In this study, we
adopted a broad definition that any fish of the Pleuronectiformes could
be labeled as 鰈魚or 比目魚 (Table 2). For that reason, even though our
barcoding analysis revealed that sushi items labeled as flatfish were
arrowtooth flounder and Greenland halibut, so that these are not the
flatfish species customers would have expected to be served, they are
still deemed to have been correctly labeled.
Traceability, the capability of keeping track of raw materials from
source to the end consumer, has been demonstrated as a good system for
preventing mislabeling and seafood fraud (Barendse et al., 2019). In
order to enhance traceability, detailed labeling information is required,
especially relating to the species constituting fishery products. In the
absence of traceability systems, consumers would easily be defrauded
and may encounter health issues, and illegal fishing activities could be
stimulated. Accordingly, the European Union has applied labeling reg­
ulations (EU No. 1379/2013) in which providing the scientific name on
fishery product labels is mandatory (see D’Amico, Armani, Gianfaldoni,
& Guidi, 2016 for a review). Previously, C.-H. Chang et al. (2016)
encouraged the Taiwanese authorities to publish a standard list of
common fish names that corresponded to scientific nomenclature.
Regrettably, that official standardized list has still not been made
available. Fish fraud is recognized as a significant issue in China, and
scientists there have also recommended generating an official list of
acceptable market names for fishes (Xiong et al., 2018; Xiong et al.,
2019; Xiong et al., 2020). In 2012, ichthyologists from China and
Taiwan collaborated to develop a Latin-Chinese dictionary of fish
names, which is available on the website of the Fish Database of Taiwan
(http://fishdb.sinica.edu.tw/). This online dictionary is frequently
updated and currently contains the Chinese names of more than 31,000
fish species. We advocate that respective authorities adopt this
Latin-Chinese dictionary of fish names as a standard to improve labeling
regulations, and also encourage those authorities to legislate for their
own labeling regulations that could be based on EU No. 1379/2013, but
which could additionally be applied to the two major groups excluded
from those EU regulations, i.e., prepared and processed products (such
12
Food Control 130 (2021) 108264
as sausages and caviar), and aquatic invertebrates (including crusta­
ceans, mollusks, and sea urchins) should no longer fall outside labeling
regulations (D’Amico et al., 2016; Tinacci et al., 2019). Moreover, these
new labeling regulations should be applied to all stages of the fishery
supply chain, including restaurants and caterers (Armani et al., 2017).
Compliance with such labeling regulations would likely result in a low
rate of seafood mislabeling (Tinacci et al., 2019).
4. Conclusion
In this study, we have demonstrated that conventional full-length
DNA barcoding can be used for the molecular identification of most
sushi samples. We report a mislabeling rate in conveyor-belt-style sushi
restaurant chains in Taiwan of ~17.36%. However, spurious reference
sequences in the BOLD database were responsible for some incorrect
DNA authentications. Fishes that are typically sensitive to fraudulent
declarations, such as tuna, are not a significant problem in Taiwan. This
may be due to the fact that the restaurant chains we sampled are high-
end establishments, so the companies are more conscious of brand
reputation, and/or it may be because mislabeling of certain fishes is less
common in locations from where the fishes are harvested (Warner,
Timme, Lowell, & Hirshfield, 2013), as is the case in Taiwan where
many sushi-destined species are caught. We found that fish roe is highly
prone to fraud. Ambiguous usage of Japanese kanji in Chinese labels, the
non-unified usage of Chinese common names, conflict between Chinese
and English common names and, most importantly, the lack of a stan­
dard list of common fish names all impede DNA barcoding efficiency. To
facilitate future investigations of food fraud to protect citizen health and
consumer rights, we advocate that the Taiwanese government estab­
lishes its own reliable and complete DNA barcoding database, publishes
a standard list of common foodstuff names that correspond to scientific
nomenclature, and legislate for complete labeling regulations to assist in
further mislabeling inspections and to deter cases of food fraud.
CRediT authorship contribution statement
Chia-Hao Chang: Conceptualization, Methodology, Writing – orig­
inal draft, preparation, Writing – review & editing. Meng-Ling Tsai:
Data curation, Software, Validation. Ting-Ting Huang: Software, Vali­
dation. Yu-Chun Wang: Supervision.
Acknowledgements
The first author was supported by a grant from the Ministry of Sci­
ence and Technology, Taiwan (MOST 109-2621-B-029-006). The au­
thors also thank Dr. Yi Ta Shao at the Institute of Marine Biology
(National Taiwan Ocean University) for offering technical assistance.
The authors are also grateful to Dr. John O’Brien for editing assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodcont.2021.108264.
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