Identification of fish species of sushi products in Hong Kong

Accepted Manuscript Identification of fish species of sushi products in Hong Kong Grace Wing-Chiu But, Hoi-Yan Wu, Pang-Chui Shaw PII:

S0956-7135(18)30545-0

DOI:

https://doi.org/10.1016/j.foodcont.2018.11.008

Reference:

JFCO 6377

To appear in:

Food Control

Received Date: 19 July 2018 Revised Date:

22 October 2018

Accepted Date: 6 November 2018

Please cite this article as: But G.W.-C., Wu H.-Y. & Shaw P.-C., Identification of fish species of sushi products in Hong Kong, Food Control (2018), doi: https://doi.org/10.1016/j.foodcont.2018.11.008. 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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Identification of fish species of sushi products in Hong Kong

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Grace Wing-Chiu Butc†, Hoi-Yan Wua,b† and Pang-Chui Shawa,b,c*

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Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China c School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

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*Corresponding author Professor Pang-Chui Shaw, Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, Institute of Chinese Medicine and School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. Tel.: +852 39431363; Fax: +852 26037246; Email address: [email protected] †

These authors contributed equally to this work.

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Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

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ACCEPTED MANUSCRIPT Abstract Sushi is a popular food product in Hong Kong and around the world. It has been

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found susceptible to fraudulent substitution or mislabeling in various countries in North America, Europe, and Asia. In Hong Kong, the hygiene aspect of sushi products is tightly regulated, with regular evaluation of bacteria content. However, less concern is put on the authenticity and labeling of sushi products. The present study represents

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a first DNA based survey for the identification of fish and fish roe used in sushi products commonly available in Hong Kong. The accuracy of the English and/or Chinese names on the product labels was also evaluated based on the common names of the identified species shown in FishBase (for English labels) and The Fish

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Database of Taiwan (for Chinese labels). Accuracy of local common names (in Chinese) were assessed individually. Ninety-three pieces of sushi, with 85 fish samples and 43 roe samples, were collected from restaurants and other retailers for 16S rRNA gene sequencing. All samples were successfully sequenced. Species identification was performed by BLAST and phylogenetic clustering. The molecular

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results were then used to verify the information declared at purchase. Out of the 128 samples, 96 samples were labeled in English and 107 samples in Chinese. The overall rate of mislabeling in English was 26.0% (25/96) and that in Chinese was 17.8% (19/107), reflecting a moderate level of mislabeling. The English labels phonetically

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translated from Japanese had a high rate of mislabeling (40%). The most commonly mislabeled sushi type was herring roe of herring sushi, most of which was identified as Mallotus villosus (capelin).

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Highlights 93 pieces of sushi, with 85 fish samples and 43 roe samples, were collected in

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Hong Kong for 16S rRNA sequencing analysis. 16S rRNA sequence can identify most fish species in sushi products at genus or

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species level. Overall rate of mislabeling in English was 26.0% and overall rate of mislabeling in Chinese was 17.8%. English labels translated from Japanese phonetically showed a high rate of mislabeling (40%).

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Keywords Sushi, DNA authentication, 16S rRNA gene; fish

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1. Introduction Mislabeling of fish has become a global epidemic (Cawthorn et al., 2012; Cutarelli et 2

ACCEPTED MANUSCRIPT al., 2014; Carvalh et al., 2015; Chang et al., 2016; Xiong et al., 2016). Reasons for mislabeling are (1) mis-identification of fish caused by similar morphological

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characteristics, (2) different names in the market for the same species, and (3) intentional adulteration (Clark, 2015). In Hong Kong, after the outbreak of more than 600 keriorrhea cases in 2006-2007, people began to pay attention to fish labelling (Ling et al., 2008). In this case, people got keriorrhea after consuming fish products,

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including sushi and sashimi, labelled as "codfish", “snowfish” or “white tuna”. These products were subsequently identified as escolar (Lepidocybium flavobrunneum) (Chong, 2007) or oil fish (Ruvettus pretiosus) (Chung, 2007; Mok, 2007). In 2016, WWF-Hong Kong (2016) collected chilled Dentex species and Plectropomus

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maculatus from major supermarket stores and found that they were Pagrus major and Ptetropomus leopardus respectively. The price of Dentex species is almost double that of P. major.

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674 retailers found that one-third of the samples were mislabeled, and that shops or restaurants selling sushi had the highest rate of mislabeling (74%) (Warner et al., 2013). Another study conducted in Los Angeles over a longer timespan (4 years, from 2012-2015) also revealed high percentage of mislabeling (47%) in sushi restaurants

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(Willette et al., 2017). In Europe, the level of substitution of sushi fish varies from country to country. Low (3.4%) to moderate (10.4%) levels of mislabeling or substitution have been found in sushi products sold in Italy (Armani et al., 2017) and the United Kingdom (Vandamme et al., 2016). However, the rate of mislabeling was

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54.5% in sushi restaurants in Belgium, mainly due to the replacement of high-priced Atlantic bluefin tuna by other low-cost tropical tuna species (Oceana, 2015). Sushi

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identification study of comparable scale is lacking in Asia. Chin and colleagues (2016) have conducted a DNA identification study on seafood products in Malaysia. Only 16

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sushi samples were involved and five of them were mislabeled. The percentage of mislabeling of sushi (31.25%) was higher than the overall rate of mislabeling of all seafood products (16%).

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and sashimi of for an adult was 4.68 g/day (Centre for Food Safety, 2010). The revenue of the three top Japanese full-service restaurants, was approximately 36.1 million Hong Kong dollars (Euromonitor, 2013). According to the Food Business

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Regulation of the Public Health and Municipal Services Ordinance (Cap. 132), sushi is identified as “food consisting of cooked and pressed rice flavoured with vinegar and garnished with other food ingredients including raw or cooked or vinegared

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In the USA, a large-scale survey involving more than 1,200 seafood samples from

In Hong Kong, sushi and sashimi are popular. The average daily consumption of sushi

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and is classified as restricted food. Hygiene aspect of sushi is strictly regulated and their bacteria content is assessed by the Centre for Food Safety (Food and Environmental Hygiene Department, 2000; Centre for Food Safety, 2014). However, less concern has been put on the labeling and the identity of sushi. Although Part V

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(Food and Drugs) of the Public Health and Municipal Services Ordinance (Cap. 132) bans any false labeling or advertisement of food and drugs, we do not have any menu labeling law in Hong Kong. There is no regulation specifying the information that must be shown on the menu for food sold in restaurants. Listing of ingredients or food

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allergens on the menu is not mandatory.

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Kong has two official languages, Chinese and English. It is common for restaurants or retailers to label or show their sushi products in common names of both languages, or in either one of them. The accuracy of Chinese and English labeling is therefore also assessed.

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Because of the susceptibility of sushi products to fraudulent substitution and the absence of similar survey in Hong Kong, we have conducted a survey to identify sushi products sold in restaurants and grocery stores by 16S rRNA sequencing. Hong

2. Materials and methods

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2.1 Sample collection and label analysis

Ninety-three samples of sushi were collected from 27 restaurants or retailers in 14 districts in Hong Kong from January 2016 to January 2017 (Table 1). Most of them

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(85 out of 93) were purchased as individual sushi samples while eight of them were from two different mixed sushi sets from the same retailer. Totally, 128 pieces

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(consisting of 85 fish samples and 43 roe samples) were analysed. After collection, the sushi was kept with ice packs before stored at -80°C on the same day. Menus were collected from the restaurants or downloaded from their websites for recording their labels in Chinese and in English. For pre-packaged products purchased from supermarkets, label information was available on the package.

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2.2 DNA extraction and PCR amplification DNA was extracted from the fish of sushi using Tissue/Cell Genomic DNA Extraction

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Kit (Biomed, Beijing, China) according to the manufacturer’s instructions. For herring samples, the fish part and the roe part were separated and rinsed with 70% ethanol before blot-dried on kimwipe paper for DNA extraction. Twenty milligrams of

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ACCEPTED MANUSCRIPT sample were used for each extraction and purified DNA was eluted in 100µL Buffer EB prewarmed to 65°C. DNA concentration was measured with NanoDrop One

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spectrophotometer (Thermo Scientific, USA). PCR was performed to amplify the 16S rRNA gene using the primers 16L1 (5’-CTGACCGTGCAAAGGTAGCGTAATCACT-3’) and 16H1 (5’-CTCCGGTCTGAACTCAGATCACGTAGG-3’) (Hedges, 1994) with a reaction

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volume of 30µL. The length of the expected amplicon is ~500 bp. Each PCR reaction mixture contained 10ng template DNA, 3µL of 10X PCR buffer, 3µL MgCl2 (25mmol/L), 2.4µL dNTP mixture (2.5mmol/L each), 1.5µL each primer (10µmol/L), and 0.2µL Taq polymerase (5U/µL). PCR was carried out in T-100 Thermo Cycler

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(Bio-rad, USA) with PCR cycles consisting of an initial denaturation at 95°C for 3 min, 35 cycles of 30 s at 95°C, 30 s at 50°C and 1 min at 72°C, followed by a final extension at 72°C for 5 min. PCR products were visualized in 1.5% agarose gel. Length of fragments was compared to 100 bp DNA marker prepared with Forever 100bp Ladder Personalizer (Seegene, USA).

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2.3 DNA sequencing, species identification by BLAST and phylogenetic analysis PCR products were purified from gel with gel extraction kit (Biomed, Beijing, China) and Sanger sequencing was performed by Tech Dragon Limited, Hong Kong. The

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sequences were manually edited with BioEdit 7.0.5 (Hall, 1999) if necessary. Species identification was performed by BLAST search at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against nucleotide sequences available on GenBank. The top-match results with a sequence similarity of 98% or higher were

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recorded. The families/subfamilies of the top-match species were searched on GenBank for their 16S sequences as references sequences. To generate the

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phylogenetic trees, when available, sequences with voucher number were downloaded (up to 5 sequences per species). For families with limited number of vouchered

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sequences, all 16S sequences of the families were downloaded. However, sequences of the same species must be at least 98% similar and outliers were discarded. Reference sequences and sample sequences of the same families were aligned using ClustalW in MEGA 7.0 software. Pairwise distances were computed using Kimura 2-paramter model (Kimura, 1980) for clustering analysis. A neighbor-joining (NJ) tree

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with Kimura 2-parameter distance was built with 1000 bootstrap re-samplings for each family.

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2.4 Evaluation of labeling accuracy As shown in Table 1, all sushi products were labeled in common names in Chinese and/or English. Scientific names were not given in all sushi products. To evaluate the

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ACCEPTED MANUSCRIPT accuracy of the common names on the labels, common names or market names in English of the identified species were obtained from FishBase

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(http://www.fishbase.de). Samples with Japanese phonetic translation (romaji) as English labels were assessed individually based on the meanings of the labels in Japanese. Chinese common names of fish were taken reference from The Fish Database of Taiwan (http://fishdb.sinica.edu.tw/), althoughother locally used common

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names and, in some cases, direct translation from Japanese names, were also accepted. Some Chinese common names are from transliteration of English common names based on similar English and Cantonese pronunciations, such as translation of tuna as “吞拿”, and herring as “希靈”. It is also common for Japanese restaurants in Hong

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Kong to use Japanese kanji characters of the fish types or sushi types as Chinese market names, such as “平目” for Paralichthys olivaceus and “筋子” for salmon roe.

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Other Chinese labels were individually assessed.

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3. Results and discussion

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3.1. Sample collection To achieve a degree of independence and to avoid repeated sampling from the same product lots of the same suppliers (Vandamme et al., 2016; Armani et al., 2017),

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samples were collected from 23 restaurants and four retailers of different brands in 14 Hong Kong districts over 13 months. Only sushi with fish or fish roes were collected as the scope of this work covers fish and fish products but not other seafood. 93 pieces of sushi belonging to 16 types of fish were collected (Table 1). A total of 128

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samples, with 85 fish samples and 43 roe samples, were obtained from the 93 sushi pieces. Most of the samples were raw but some of them were cooked (5 out of 128),

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marinated (13 out of 128) or with sauce or mayonnaise added (25 out of 128). Among the 93 sushi pieces, 67 sushi pieces were labeled in both Chinese and English, seven

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were in English only, and 18 were in Chinese only. In total, 85 samples had Chinese labels and 74 samples had English labels. One piece of sushi (Z8) was unlabeled and three had unclear labels. These include: (1) Sample A2 was labeled as “red fish”. (2) Sample U2 was labeled “澗白（壽）” in Chinese or Japanese and “Akirabeni” in

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English. The exact meaning of these labels was unknown. (3) Sample L3 was labeled as “白玉豚壽司” and the meaning was obscure. One “crab roe” sushi (Z6) was

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collected from a mixed sushi set. It was analyzed together with other fish sushi. Twenty-one pieces of “flower of love”, a kind of sushi originated in Hong Kong

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(Apple Daily, 2005), were collected. It consists of raw salmon and roes (usually salmon roes or flying fish roes) rolled into the shape of a flower with rice. Its fish part would presumably be salmon. The identity of roes was usually not labelled. Fig. 1

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ACCEPTED MANUSCRIPT shows the images of typical “flowers of love” with different kind of roes and the images of the four sushi products with unclear labeling of fish type.

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According to the Food and Drugs (Composition and Labelling) Regulations (Cap. 132W), labelling of prepackaged food is required to include (1) name of the food, (2) list of ingredients, (3) indication of durability, (4) special conditions for storage or instruction for use, (5) amount (count, weight or volume), and (6) name and address

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of manufacturer or packer in English or Chinese or both languages. Two prepackaged mixed sushi sets were collected from the same supermarket. One consists of sample “Tuna” (Z2), “Snapper” (Z3), “Sea eel” (Z4), “Salmon” (Z5), and “Crab roe” (Z6) and the another consists of sample “Eel tail” (Z7), “Salmon” (Z9), and Z8, which was

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unlabelled. Both sets had provided all six pieces of information required by the labelling of prepackaged food. However, the labels on both sushi sets were incomplete (Z8) or incorrect (Z6). They were analyzed together with other sushi.

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The marker selected in this study was 16S rRNA gene from mitochondrial DNA (mtDNA). Sequences of 16s rRNA have been successfully used for species identification of fish in canned pet food (Armani et al., 2015), dried fish maws (Zeng et al., 2018) and dried scallop adductor muscle (Wen et al., 2017). A set of universal

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primers, 16Sar-5’ and 16Sbr-3’, was used to amplify a DNA fragment of around 600 bp from 92 out of 94 fish families (Ivanova et al., 2006). However, mediocre successful rate of amplification and sequencing has been reported for this universal primer set on dried fish maws, with only 36 out of 79 specimens successfully

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sequenced (Tuuli et al., 2016). The primer set used in this study, 16H1 and 16L1, amplifies the same region of 16S rRNA gene but with a shorter amplicon (about 500

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bp). Most sushi pieces contained a single piece of fish. For sushi containing both fish and roes, as in “flower of love” and herring sushi, DNA of the fish part and the roes

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were extracted and sequenced separately with distinct sample codes. The concerned 16S rRNA DNA fragment was successfully amplified and sequenced without cloning from the 85 fish samples and 43 roe samples. Cooking, marination and presence of sauce or mayonnaise did not affect amplification and sequencing.

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3.3 Sequencing and molecular identification All the sequences obtained gave a 98%-100% similarity to their corresponding top-match sequences from BLAST (Accession numbers with the highest BLAST score

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shown in Table 1; Accessions with 98% similarity or higher shown in Table S1). Tree-based method was adopted to verify the species identified by BLAST as well as to evaluate the discriminatory power of the 16S rRNA gene fragment in different

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3.2. DNA extraction and PCR amplification

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ACCEPTED MANUSCRIPT groups of fish species. Fourteen neighbor-joining dendrograms (Fig. S1 – S14) were constructed using the Kimura 2-parameter distance model. Most samples were

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identified up to genus- or even species-level, as listed in Table 2. For samples identified at species-level with >80% bootstrap values, a good agreement between the matched species from BLAST and that from phylogenetic clustering has been achieved. From Fig. S1a shows clustering of various Oncorhynchus species was not

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well supported, with bootstrap values ranging from 48% to 87%. Distance analysis was carried out again with reference sequences from Oncorhynchus species only (Fig. S1b). This time, species-level clustering with >80% bootstrap values for most clusters was resulted, supporting the matching of salmon roe samples to O. keta, O. kisutch

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and O. gorbuscha. Samples belonging to Cheilopogon (Fig. S2), Clupea (Fig. S3), Thunnus (Fig. S8), Sebastes sp. (Fig. S14), Atheresthes or Pseudopleuronectes (Fig. S6) genus could not be further resolved to single species using this partial 16S fragment.Other research groups also identified tuna and flying fish at genus level only based on the COI

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sequences,as there was little interspecific divergence for these congeneric species (Vandamme et al., 2016; Armani et al., 2017). Identity of sample T1 (flounder) (Fig. S5), A1 and B7 (tilapia) (Fig. S10) could not be determined from the phylogenetic trees. The assessment of substitution or mislabeling, however, was not much affected

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as the umbrella terms, such as “tuna”, “flying fish” and “tilapia”, were used as labels. . 3.4 Evaluation of labeling information This study is the first systematic authenticity survey of sushi products sold in Hong

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Kong. Because of the absence of official species name-to-market name guideline or list, like The Seafood List of the U.S. Food and Drug Administration, in Hong Kong,

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we searched for the English common names of the fish species identified from the FishBase to assess the accuracy of labels. Chinese names were searched from The

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Fish Database of Taiwan but locally used Chinese common names were assessed individually. It is not straightforward to assess the accuracy of the English and Chinese labels. Without any menu labeling regulations, it is not mandatory to indicate the ingredients in the food name or to provide an ingredient list on the menus of restaurants and takeouts. Only prepackaged sushi products are required to include a

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list of ingredients on the food labels as stated in Food and Drugs (Composition and Labelling) Regulations (Cap. 132W). However, prepackaged sushi products only constituted a small portion in this study. Therefore, we had to first determine whether

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the label on the sushi or from the menu contains any information about the type of fish. This may be complicated as (1) some English labels are phonetic translated from Japanese and some Chinese labels are from Japanese kanji characters; (2) some

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Among the “flowers of love” we collected, thirteen had the same meaning in Chinese. For these samples, the type of roes used was not specified. Only two samples had clearly specifying salmon roe (三文魚籽) in their Chinese labels. One sample had

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labeled crab roe (蟹籽) and one sample had labeled capelin roe (多春魚籽). Four

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samples had the Chinese meaning of “parent-and-children”, in their Chinese labels (Table 1). For these Chinese labels, labeling accuracy could be assessed. It is common to find different or slightly different Chinese characters with the same pronounciation for the same kind of fish. Hence, both “鱆紅魚” and “章紅” are

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accepted as correct Chinese labels of greater amberjack (Seriola dumerili). Seven out of nine flounder sushi samples were labeled as “左口魚” (meaning left-mouth fish),

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but five of them belong to family Pleuronectidae (righteye flounders). The common name “左口魚” are used locally in Hong Kong and nearby regions to refer to

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flounders or flatfish. Therefore, we regarded such labeling of flounder samples, even

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for righteye flounder, as correct. However, for ensuring the safety and rights of consumers, use of separate common names for lefteye and righteye flounders should be encouraged.

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3.5 Sushi mislabeling in Hong Kong In this study, the overall rates of mislabeling in English and in Chinese were 25/96 (26.0%) and 19/107 (17.8%), respectively. This level of misdescription is moderate, compared to the low mislabeling rate in Italy (3.4%) and the UK (10.4%) (Armani et

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al., 2017; Vandamme et al., 2016) and the high rate (74%) in North America (Warner et al., 2013). Out of the 84 English labels, 10 of them were in romaji, the phonetic

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translation of Japanese into alphabets. The rate of wrong description in this kind of labels was 40% (4/10), reflecting a common misuse of Romanized Japanese as

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English label in Hong Kong. Salmon is the most consumed fish type in sashimi and sushi in Hong Kong. It was recently reported that one-third of salmon sold in China was actually rainbow trout (Oncorhynchus mykiss) raised in freshwater fish farm in Qinghai province (Yan, 2018). In Japan, rainbow trout was found to be used as a substitute for salmon and

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labelled as ‘salmon trout’ instead of ‘rainbow trout’ (Sone & Nortvedt, 2009). Since rainbow trouts infected by parasites were found from time to time (Torres et al., 2002; Unger & Palm, 2017), there was public concern on the authenticity of salmon sold in

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Hong Kong. In this study, all salmon sushi products were correctly labeled and no trout had been found. The most mislabeled fish type was herring roe, with a mislabeling rate of 92.9% in

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ACCEPTED MANUSCRIPT English and 83.4% in Chinese. These “herring roe” samples were actually Mallotus villosus (capelin). In Hong Kong, herring sushi usually contains fish and roe together

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on the rice ball. The fish part was correctly labelled in all samples. Only one out of thirteen herring samples which were labelled as “herring” contained herring roe and only one out of twelve herring samples which were found to contain capelin roe clearly labelled “herring with capelin roe.” This substitution is probably economically

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motivated, as herring is more expensive than capelin in the market. The retail price of seasoned herring roe can be about seven times that of seasoned capelin roe in Hong Kong (seasoned herring roe – HKD 263 per 180g; seasoned capelin roe – HKD 100 per 500g, Marche HK). In the USA, the retail price of herring roe cavier is about 2.8

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times that of capelin roe cavier ($6.56 vs $2.30 per oz, gourmet food store Marky’s). As many fish species have similar appearance, taste and texture, it is relatively easy to substitute species of high commercial value with those of lower commercial value (Cawthorn et al., 2012). The size of herring roe is very similar to capelin roe (Bledsoe et al., 2010). In Japan, salted mixture of capelin roe and fish surimi have been

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consumed as substitute of herring roe product (Gomyo & Furukawa, 2005). Three out of four English labels of greater amberjack were incorrect. Two of them were labeled as rudderfish. One of the two flying fish roe samples was wrongly labeled as crab roe. The labeling of flying fish roe as crab roe is common in Hong

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Kong and is even well known to the public. Yellowtail, yellowfin tuna and bluefin tuna had a high rate of mislabeling in the West (Willette et al., 2017; Oceana, 2015). We found that all tuna samples and most yellowtail samples (10/11) were correctly labeled in Hong Kong.

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Four samples contained labels that did not indicate the fish types. One was labeled as “Oilfish” in English and we identified it as Lepidocybium flavobrunneum (escolar).

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Escolar had been found as substitution of other fish with high commercial value (Giusti et al., 2016). The wax ester of oilfish and escolar can cause keriorrhea. After

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the oilfish outbreak, the Centre of Food Safety of Hong Kong government has reached consensus with the industry “to suspend import and sale of oilfish and to stop using oilfish for catering purpose” and “to destroy their remaining oilfish stock” in 2007 (Centre for Food Safety, 2007). However, the sale of oilfish/escolar has not been banned and oilfish is still available in the market. Such unclear, misleading labels fail

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to provide sufficient information to the consumers and would create a potential health hazard to the public. Although Food and Drugs (Composition and Labelling) Regulations (Cap. 132, section 55) and Trade Descriptions Ordinance (Cap. 362) are

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in place to make sure the products are not wrongly labeled and no false information is put on the labels/packages, it is not illegal to name the products with names of unclear meaning, as in the case for sample U2.

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In this work, the genetic identification of fish samples from sushi products in Hong Kong was performed by sequencing of 16S rRNA fragment with universal primers. Successful rate of amplification and sequencing using the primer sets 16H1 and 16L1 was 100%. Except for the three samples that cannot be resolved by phylogenetic

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clustering, the rest can be identified at least up to genus level. The rate of mislabeling was moderate (26.0% for English labels and 17.8% for Chinese labels). Most popular fish types in sushi and sashimi, including salmon, tuna, eel and yellowtail, were correctly labeled. Herring roe of herring sushi was found to have the highest rate of

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misdescription and was heavily substituted by capelin roe. Oilfish was identified in one sushi sample, which was correctly labeled as “Oilfish” in English but unclearly labeled in Chinese. Our work shows that regulation on the labelling of Sushi products in Hong Kong is needed.

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Acknowledgements We thank Mr Ka-Lok Wong for technical assistance and Ms Natalie Wing-Sum Yu for collecting some of the samples from different restaurants in Hong Kong. This project was initiated as a practical session for the Molecular Biology and Recombinant DNA

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Laboratory course of the Biochemistry Programme of The Chinese University of Hong Kong in 2016.

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Figure captions

Fig. 1. Some of the sushi products in this study. (A) “Flower of love” sushi with different roes. (B) Sushi products with unclear fish type on labels. Fig. 2. Amplification of the 16S rRNA regions from different sushi products. Sizes of all amplicons are ~ 500 bp. m = 100bp DNA marker. Lanes A1-Z9, A’1-A’2 = Samples A1-Z9 and A’1-A’2 from Table 1. N = no template control.

575 576

Fig. 3. Percentage of mislabeling of different fish types in Chinese and in English.

577 578

The number at the end of each bar is the number of mislabeled samples out of the number of labeled samples (in Chinese or English) collected for that type of fish. N/A

579 580 581 582

indicates that all samples were not labeled in that language.

583 584 585 586

Fig S1a. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

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the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the

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Captions for supplementary figures

sequences obtained from salmon (n = 23) and salmon roe (n = 9) products. The distance analysis was computed using the Kimura two parameter genetic distance involving 84 nucleotide sequences (n = 32 from commercial products and 52 reference sequences retrieved from GenBank) with 247 positions in

branches. Reference sequence: 16S sequences of Salmonidae with voucher specimens in GenBank and all 16S 16

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sequences of Oncorhynchus without voucher specimens from GenBank (with “NV” at the end of

593 594 595 596

Fig S1b. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

597 598 599 600

Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

601 602 603

Fig S2. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

604 605 606 607

involving 44 nucleotide sequences (n = 14 from commercial products and 30 reference sequences

608 609 610 611

samples” that were not directly from the organisms.

612 613 614 615 616 617

of herring sushi (n = 1) products. The distance analysis was computed using the Kimura two parameter

.

sequences obtained from salmon roe (n = 9) products. The distance analysis was computed using the Kimura two parameter genetic distance involving 36 nucleotide sequences (n = 9 from commercial

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products and 27 reference sequences retrieved from GenBank) with 293 positions in the final dataset.

Reference sequence: 16S sequences of Oncorhynchus without voucher specimens from GenBank (with “NV” at the end of accession number)

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.

sequences obtained from roes of “flower of love” (n = 12), crab roe (n = 1) and flying fish roe (n = 1)

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products. The distance analysis was computed using the Kimura two parameter genetic distance

retrieved from GenBank) with 374 positions in the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

Reference sequence: 16S sequences of Exocoetidae in GenBank, excluding the “environmental

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Fig S3. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA sequences obtained from roes of “flower of love” (n = 3), fish parts of herring sushi (n = 14) and roes

genetic distance involving 170 nucleotide sequences (n = 18 from commercial products and 152

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reference sequences retrieved from GenBank) with 191 positions in the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: Selected 16S sequences of Clupeidae with voucher specimens in GenBank. Up to five sequences for each species.

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accession number)

Fig S4. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA sequences obtained from roes of “flower of love” (n = 1), roes of herring sushi (n = 12) and roes of

621 622 623 624

capelin (n = 1) products. The distance analysis was computed using the Kimura two parameter genetic

625 626 627

Reference sequence: 16S sequences of Salangidae in GenBank, excluding the 12 sequences that are too

distance involving 107 nucleotide sequences (n = 14 from commercial products and 93 reference sequences retrieved from GenBank) with 299 positions in the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

short (<250 bp) or not covering the same regions of our amplicons.

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Fig S5. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

631 632 633 634

the Kimura two parameter genetic distance involving 82 nucleotide sequences (n = 21 from

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Reference sequence: 16S sequences of Carangidae with voucher specimens in GenBank, excluding 5

639 640 641

Fig S6. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

642 643 644 645

and 153 reference sequences retrieved from GenBank) with 209 positions in the final dataset. Bootstrap

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number)

650 651 652 653 654 655

two parameter genetic distance involving 41 nucleotide sequences (n = 2 from commercial products

656 657 658

Fig S8. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

659 660 661 662

127 nucleotide sequences (n = 7 from commercial products and 120 reference sequences retrieved from

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sequences of Thunnus without voucher specimens (with “NV” at the end of accession number).

sequences obtained from greater amberjack sushi(n = 7), yellowtail sushi (n = 11), flounder sushi (n = 1) and one sushi without specifying fish type on its label (U2). The distance analysis was computed using

commercial products and 61 reference sequences retrieved from GenBank) with 281 positions in the

branches.

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final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the

“environmental sample” sequences and 9 Trachinotus sequences that were matched to Larimichthys

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crocea in BLAST search and were probably mis-identified.

sequences obtained from flounder sushi (n = 6). The distance analysis was computed using the Kimura

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two parameter genetic distance involving 159 nucleotide sequences (n = 6 from commercial products

values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: 16S sequences of Pleuronectidae with voucher specimens in GenBank and Pseudopleuronnectes sequences without voucher specimens (with “NV” at the end of accession

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Fig S7. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA sequences obtained from flounder sushi (n = 2). The distance analysis was computed using the Kimura

and 39 reference sequences retrieved from GenBank) with 196 positions in the final dataset. Bootstrap

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values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: Vouchered 16S sequences of Paralichthyidae in GenBank and 16S sequences of

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Paralichthys without voucher specimens (with “NV” at the end of accession number).

sequences obtained from tuna sushi (n = 5), mackerel sushi (n = 2) and one grilled fish sushi (sample Z1). The distance analysis was computed using the Kimura two parameter genetic distance involving

GenBank) with 361 positions in the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: 16S sequences of Scombridae with voucher specimens in GenBank and all 16S

Fig S9. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA 18

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sequences obtained from swordfish sushi (n = 3). The distance analysis was computed using the

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Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

673 674 675 676

sequences obtained from Tilapia sushi (n = 2). The distance analysis was computed using the Kimura

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Reference sequence: 16S sequences of subfamily Pseudocreniabrinae in GenBank.

680 681 682 683

sequences obtained from Japanese eel sushi (n = 2). The distance analysis was computed using the

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Reference sequence: 16S sequences of Angiulla with voucher specimens in GenBank.

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the Kimura two parameter genetic distance involving 37 nucleotide sequences (n = 1 from commercial

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Fig S13. Condensed distance tree inferred using the Neighbor-Joining method for the analysis of partial

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from commercial products and 158 reference sequences retrieved from GenBank) with 427 positions in

701 702 703

specimens in GenBank.

Kimura two parameter genetic distance involving 26 nucleotide sequences (n = 3 from commercial products and 23 reference sequences retrieved from GenBank) with 394 positions in the final dataset.

Reference sequence: 16S sequences of Order Isotiophoriformes in GenBank.

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Fig S10. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

two parameter genetic distance involving 74 nucleotide sequences (n = 2 from commercial products

and 72 reference sequences retrieved from GenBank) with 323 positions in the final dataset. Bootstrap

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values (BP) > 50% obtained from 1000 replicates are shown below the branches.

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Fig S11. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA

Kimura two parameter genetic distance involving 41 nucleotide sequences (n = 2 from commercial products and 39 reference sequences retrieved from GenBank) with 229 positions in the final dataset. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

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Fig S12. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA sequences obtained from oilfish sushi (n = 1, sample L3). The distance analysis was computed using

products and 36 reference sequences retrieved from GenBank) with 376 positions in the final dataset.

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Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: Selected 16S sequences of Gempylidae (up to five sequences per species) with

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voucher specimens in GenBank.

16S rRNA sequences obtained from red fish sushi (n = 1, sample A2). The distance analysis was computed using the Kimura two parameter genetic distance involving 159 nucleotide sequences (n = 1

the final dataset at 70% cutoff value. Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches. Reference sequence: Selected 16S sequences of Sebastidae (1-2 sequences per species) with voucher

Fig S14. Distance tree inferred using the Neighbor-Joining method for the analysis of partial 16S rRNA 19

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sequences obtained from red fish sushi (n = 1, sample Z8). The distance analysis was computed using

707 708 709 710

Bootstrap values (BP) > 50% obtained from 1000 replicates are shown below the branches.

the Kimura two parameter genetic distance involving 58 nucleotide sequences (n = 1 from commercial products and 57 reference sequences retrieved from GenBank) with 389 positions in the final dataset.

Reference sequence: Selected 16S sequences of Sparidae (up to five sequences per species) with voucher specimens in GenBank and all Pagrus 16S sequences without voucher specimens (with “NV”

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at the end of accession number).

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Table 1. Label information and identification results of sushi products

English label

Part

Matched % Species matched by BLAST accession from Identity BLAST

Common name (FishBase)

Chinese name (The Fish Database from Taiwan)

Species identified by phylogenetic analysis

Chinese English Remark label label accuracy accuracy

Salmon sushi Z5 a N/A

Salmon

Fish

KF792729.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

N/A

correct

Z9 b

Salmon

Fish

KF792729.1

99%

Salmo salar

Atlantic salmon

Salmo salar Mislabeling

N/A N/A

correct 0/2

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

99% 99%

Oncorhynchus keta Salmo salar

Chum salmon Atlantic salmon

鉤吻鮭(大麻哈魚) 安大略鱒(大西洋鮭)

Oncorhynchus keta Salmo salar

correct correct

correct correct

98-99% 99% 99% 99%

Mallotus villosus Salmo salar Cheilopogon antoncichi Salmo salar

Capelin Atlantic salmon Spotfin flyingfish Atlantic salmon

毛鱗魚 安大略鱒(大西洋鮭) 安東鬚唇飛魚 安大略鱒(大西洋鮭)

Mallotus villosus Salmo salar Cheilopogon sp. Salmo salar

N/A correct N/A correct

wrong N/A N/A correct

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

N/A

correct

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

親子卷

Salmon & salmon roe roll

F6 G3

花之戀

Flower of love

G4 H3 H2 I3

花之戀

N/A

花之戀 (三文 N/A 魚 多春魚籽)

I4 J1

花之戀

N/A

J2 K3

花之戀

Flower of love

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安大略鱒(大西洋鮭)

Fish

KJ128886.1

99%

Salmo salar

Roe Fish

KJ010737.1 KF792729.1

99% 100%

Oncorhynchus kisutch Salmo salar

Coho salmon Atlantic salmon

銀鉤吻鮭(銀大麻哈魚) 安大略鱒(大西洋鮭)

Oncorhynchus kisutch Salmo salar

correct correct

correct correct

Roe

KY067950.1

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

N/A

N/A

Fish

KF792729.1

100%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

N/A

Roe Fish

KC193776.1 KF792729.1

99% 100%

Clupea harengus Salmo salar

Atlantic herring Atlantic salmon

大西洋鯡 安大略鱒(大西洋鮭)

Clupea sp. Salmo salar

N/A correct

N/A N/A

Roe

AB444863.1

99%

Cheilopogon antoncichi

Spotfin flyingfish

安東鬚唇飛魚

Cheilopogon sp.

wrong

N/A

Fish

KF792729.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

N/A

Roe

AB444863.1

99%

Cheilopogon antoncichi

Spotfin flyingfish

安東鬚唇飛魚

Cheilopogon sp.

N/A

N/A

Fish

KF792729.1

98%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

EP

F5

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Salmon and salmon roe roll / Flower of Love (with mayonnaise) B4 三文魚籽花之 Salmon & salmon Fish KJ128886.1 戀 roe B5 Roe AP010773.1 C2 花之戀 Salmon and flying Fish KJ128886.1 fish roe C3 Roe GU233808.1 D1 花之戀 N/A Fish KR476892.1 D2 Roe AB444863.1 E5 花之戀 Salmon and flying Fish KJ128886.1 fish roe E6 Roe KY067950.1

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N/A

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Code Chinese label

*marinated

*marinated

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KY067950.1

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

N/A

N/A

Fish

KF792729.1

98%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe Fish

KC193776.1 KF792729.1

100% 98%

Clupea harengus Salmo salar

Atlantic herring Atlantic salmon

大西洋鯡 安大略鱒(大西洋鮭)

Clupea sp. Salmo salar

N/A correct

N/A N/A

Roe

AB444863.1

99%

Cheilopogon antoncichi

Spotfin flyingfish

安東鬚唇飛魚

Cheilopogon sp.

N/A

N/A

Fish

KF792729.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe Fish Roe

AP010773.1 KR476892.1 AP010773.1

99% 99% 99%

Oncorhynchus keta Salmo salar Oncorhynchus keta

Chum salmon Atlantic salmon Chum salmon

鉤吻鮭(大麻哈魚) 安大略鱒(大西洋鮭) 鉤吻鮭(大麻哈魚)

Oncorhynchus keta Salmo salar Oncorhynchus keta

correct correct correct

correct correct correct

Flower salmon sushi Fish Roe

KJ128886.1 AB444863.1

98% 99%

Salmo salar Cheilopogon antoncichi

Atlantic salmon Spotfin flyingfish

安大略鱒(大西洋鮭) 安東鬚唇飛魚

Salmo salar Cheilopogon sp.

correct N/A

correct N/A

Fish

KF792729.1

100%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe

KY067950.1

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

N/A

N/A

Fish

KJ128886.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe Fish

AB246179.1 KJ128886.1

99% 99%

Clupea pallasii Salmo salar

Pacific herring Atlantic salmon

太平洋鯡 安大略鱒(大西洋鮭)

Clupea sp. Salmo salar

N/A correct

wrong correct

Roe

AP010773.1

99%

Oncorhynchus keta

Chum salmon

鉤吻鮭(大麻哈魚)

Oncorhynchus keta

correct

correct

Fish

KJ128886.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe

KY067950.1

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

wrong

correct

N1

親子花之戀

Salmon roe with salmon

N2 S2 S3

三文親子戀

Salmon & roe roll

T2 T3

花之戀

U5

花之戀（壽） Salmon flower

U6

V3 V4 W3

花之戀

Salmon roes sushi

三文魚籽花之 Salmon w/ salmon 戀 roe roll

W4 W5

三文魚蟹籽花 Salmon w/ flying 之戀 rish roll

W6

SC

M2

M AN U

花之戀/ 三文 N/A 魚剌身

TE D

Hana Maki (Salmon)

EP

L5 M1

花之戀

AC C

L4

RI PT

Roe

K4

X4 X5

花之戀

N/A

Fish Roe

KJ128886.1 KY067950.1

99% 99%

Salmo salar Cheilopogon cyanopterus

Atlantic salmon Margined flyingfish

安大略鱒(大西洋鮭) 青翼鬚唇飛魚

Salmo salar Cheilopogon sp.

correct N/A

N/A N/A

Y2

親子卷

Salmon & salmon roe roll

Fish

KF792729.1

99%

Salmo salar

Atlantic salmon

安大略鱒(大西洋鮭)

Salmo salar

correct

correct

Roe

KJ010737.1

99%

Oncorhynchus kisutch

Coho salmon

銀鉤吻鮭(銀大麻哈魚)

Oncorhynchus kisutch

correct

correct

Mislabeling Mislabeling

Fish Roe

0/21 2/8 (25%)

0/15 2/10 (20%)

Y3

*marinated *marinated

*marinated

*marinated

ACCEPTED MANUSCRIPT

Roe

AB898738.1

99

Oncorhynchus gorbuscha

Pink salmon

細鱗鉤吻鮭(細鱗大麻哈魚) Oncorhynchus gorbuscha

correct

correct

*marinated

Salmon roe N/A Salmon roe sushi

Roe Roe Roe

AB898738.1 AP010773.1 AP010773.1

99% 99% 99%

Oncorhynchus gorbuscha Oncorhynchus keta Oncorhynchus keta

Pink salmon Chum salmon Chum salmon

細鱗鉤吻鮭(細鱗大麻哈魚) Oncorhynchus gorbuscha 鉤吻鮭(大麻哈魚) Oncorhynchus keta 鉤吻鮭(大麻哈魚) Oncorhynchus keta

correct correct correct

correct N/A correct

*marinated *marinated *marinated

Roe

AP010773.1

99%

Oncorhynchus keta

Chum salmon

鉤吻鮭(大麻哈魚)

Oncorhynchus keta

correct

correct

*marinated

Mislabeling

0/5

0/4

杜氏鰤(高體鰤)

Seriola dumerili

correct

N/A

A3 D4 V2

三文魚籽 三文魚子 三文魚籽

A’2

三文魚子壽司 Salmon roe

Greater amberjack sushi D3 鱆紅魚 N/A

Fish

AB517558.

99

Seriola dumerili

Greater amberjack

RI PT

Salmon roe

SC

Salmon roe sushi E2 筋子

鱆紅魚 鱆紅魚

Rudderfish N/A

Fish Fish

AB517558.1 AB517558.1

99% 99%

Seriola dumerili Seriola dumerili

Greater amberjack Greater amberjack

杜氏鰤(高體鰤) 杜氏鰤(高體鰤)

Seriola dumerili Seriola dumerili

correct correct

wrong N/A

L1

鱆紅魚壽司

Rudderfish

Fish

AB517558.1

99%

Seriola dumerili

Greater amberjack

杜氏鰤(高體鰤)

Seriola dumerili

correct

wrong

N3

鱆紅魚壽司

Red snapper

Fish

AB517558.1

99%

Seriola dumerili

Greater amberjack

杜氏鰤(高體鰤)

Seriola dumerili

correct

wrong

O2

章紅

Amberjack

Fish

AB517558.1

99%

Seriola dumerili

Greater amberjack

杜氏鰤(高體鰤)

Seriola dumerili

correct

correct

I1

鱆紅魚

N/A

Fish

AB517556.1

100%

Seriola quinqueradiata

五條鰤(油甘魚)

Seriola quinqueradiata

wrong

N/A

Mislabeling

1/7 3/4 (14.3%) (75%)

TE D

Yellowtail sushi C5 鰤魚 (*油甘 魚) E1 油甘魚

M AN U

G2 J3

Japanese amberjack

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

Yellowtail

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

R1

油甘魚-原味和 Yellow tail Fish 風 original 油甘魚壽司 Hamachi (Japanese Fish amberjack) 油甘魚壽司 Hamachi Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

S1

長油甘魚壽司 Long yellow tail

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

U4

油甘魚（壽） Yellowtail

Fish

AB517556.1

100%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

K1

AC C

F2

EP

Hamachi

ACCEPTED MANUSCRIPT

油甘魚

Yellowtail

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

X3

油金魚

N/A

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

N/A

Y5

油甘魚壽司

Yellow tail

Fish

AB517556.1

100%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

A’1

油甘魚壽司

Hamachi

Fish

AB517556.1

99%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

correct

correct

Z3 a

N/A

Snapper

Fish

AB517556.1

100%

Seriola quinqueradiata

Japanese amberjack

五條鰤(油甘魚)

Seriola quinqueradiata

N/A

wrong

Mislabeling

0/11

1/11 (9.1%)

Clupea sp.

correct

correct

*processed

SC

RI PT

W2

Herring sushi A5 希靈魚

Pickled herring

Fish

AB246179.1

100%

Clupea pallasii

Pacific herring

A6 B8

希靈魚

Herring fish

Roe Fish

GU233808.1 AB246179.1

99% 100%

Mallotus villosus Clupea pallasii

Capelin Pacific herring

毛鱗魚 太平洋鯡

Mallotus villosus Clupea sp.

wrong correct

wrong correct

*processed

B9 C1

希靈魚

GU233808.1 AB246179.1

99% 99%

Mallotus villosus Clupea pallasii

Capelin Pacific herring

毛鱗魚 太平洋鯡

Mallotus villosus Clupea sp.

wrong correct

wrong correct

*processed

C6 G1

希靈魚

Roe Herring with capelin Fish roe Roe Herring Fish

GU233808.1 KC193720.1

99% 99%

Mallotus villosus Clupea harengus

Capelin Atlantic herring

毛鱗魚 大西洋鯡

Mallotus villosus Clupea sp.

wrong correct

correct correct

*processed

G5 H1

希靈魚

Herring

Roe Fish

GU233808.1 KC193769.1

99% 99%

Mallotus villosus Clupea harengus

Capelin Atlantic herring

毛鱗魚 大西洋鯡

Mallotus villosus Clupea sp.

wrong correct

wrong correct

*processed

希靈魚壽司

Herring

Roe Fish Roe

GU233808.1 KJ128741.1 GU233808.1

98% 99% 99%

Mallotus villosus Clupea harengus Mallotus villosus

Capelin Atlantic herring Capelin

毛鱗魚 大西洋鯡 毛鱗魚

Mallotus villosus Clupea sp. Mallotus villosus

wrong correct wrong

wrong correct wrong

M3 M4

希靈魚剌身

Nishin

Fish Roe

KJ128741.1 GU233808.1

99% 98%

Clupea harengus Mallotus villosus

Atlantic herring Capelin

大西洋鯡 毛鱗魚

Clupea sp. Mallotus villosus

correct wrong

correct wrong

*processed

O1

希靈魚籽壽司 Hokkai Nishin

Fish

KJ128741.1

100%

Clupea harengus

Atlantic herring

大西洋鯡

Clupea sp.

correct

correct

*processed

Roe

GU233808.1

98%

Mallotus villosus

Capelin

毛鱗魚

Mallotus villosus

wrong

wrong

P1 P3

希靈魚

N/A

Fish Roe

KJ128741.1 GU233808.1

100% 98%

Clupea harengus Mallotus villosus

Atlantic herring Capelin

大西洋鯡 毛鱗魚

Clupea sp. Mallotus villosus

correct wrong

N/A N/A

*processed

Q2 Q3

希靈魚

Herring roe sushi

Fish Roe

KC193720.1 GU233808.1

99% 99%

Clupea harengu Mallotus villosus

Atlantic herring Capelin

大西洋鯡 毛鱗魚

Clupea sp Mallotus villosus

correct wrong

correct wrong

*processed

U3 U7

希靈魚（壽） Herring

Fish Roe

KJ128741.1 GU233808.1

99% 98%

Clupea harengus Mallotus villosus

Atlantic herring Capelin

大西洋鯡 毛鱗魚

Clupea sp. Mallotus villosus

correct wrong

correct wrong

*processed

V1

希靈魚

Fish

KJ128741.1

100%

Clupea harengus

Atlantic herring

大西洋鯡

Clupea sp.

correct

correct

*processed

O3

Herring sushi

M AN U

TE D

EP

AC C

H4 L2 L6

太平洋鯡

*processed

ACCEPTED MANUSCRIPT

V5

Roe

KJ128741.1

99%

Clupea harengus

Atlantic herring

大西洋鯡

Clupea sp.

correct

correct

Fish

AB246179.1

99%

Clupea pallasii

Pacific herring

太平洋鯡

Clupea sp.

correct

N/A

N/A

Roe

GU233808.1

98%

Mallotus villosus

Capelin

毛鱗魚

Mallotus villosus

wrong

N/A

希靈魚

Herring w/ capelin Fish

AB246179.1

99%

Clupea pallasii

Pacific herring

太平洋鯡

Clupea sp.

correct

correct

Roe

AB444863.1

99%

Cheilopogon antoncichi

Spotfin flyingfish

安東鬚唇飛魚 Mislabeling Mislabeling

Cheilopogon sp. Fish Roe

wrong 0/14 13/14 (92.9%)

wrong 0/12 10/12 (83.3%)

Flounder sushi B6 左口魚裙邊

Fish

HE863808.1

99%

Atheresthes evermanni

Kamchatka flounder

亞洲箭齒鰈

Atheresthes sp.

correct

wrong

E4

Olive flounder muscle 左口魚裙邊 Olive flounder muscle 左口魚邊壽司 Engawa 左口魚邊 N/A 左口魚邊 N/A 平目魚 Japanese flounder 平目魚邊（壽）Flounder skirt

Fish

FJ870391.1

99%

Atheresthes evermanni

Kamchatka flounder

亞洲箭齒鰈

Atheresthes sp.

correct

wrong

Fish Fish Fish Fish Fish

HE863808.1 FJ870391.1 FJ870391.1 AB028664.1 AB517556.1

100% 99% 98% 99% 99%

Atheresthes evermanni Atheresthes evermanni Atheresthes evermanni Paralichthys olivaceus Seriola quinqueradiata

Kamchatka flounder Kamchatka flounder Kamchatka flounder Bastard halibut Japanese amberjack

亞洲箭齒鰈 亞洲箭齒鰈 亞洲箭齒鰈 牙鮃 五條鰤(油甘魚)

Atheresthes sp. Atheresthes sp. Atheresthes sp. Seriola quinqueradiata

correct correct correct correct wrong

correct N/A N/A correct wrong

平目魚壽司 Japanese flounder 左口魚裙邊壽 N/A 司 左口魚邊壽司 Flatfish sushi

Fish Fish

AB028664.1 KX234659.1

99% 98%

Paralichthys olivaceus Pseudocaranx wrighti

Bastard halibut Skipjack trevally

牙鮃 沙擬鰺

Paralichthys olivaceus Not determined

correct correct

correct N/A

Fish

KJ936354.1

99%

Pseudopleuronectes obscurus

N/A

黑光鰈

Pseudopleuronectes sp.

correct

correct

Mislabeling

1/10 (10%)

3/7 (42.9%)

Q1

Tuna sushi B1 吞拿魚 E3 辛辣長鰭吞拿 魚 F1 備長吞拿魚

SC

M AN U

Y4 T1

Albacore Albacore

長鰭鮪(長鰭金槍魚) 長鰭鮪(長鰭金槍魚)

Thunnus sp. Thunnus sp.

correct correct

correct correct

98-99% Thunnus alalunga

Albacore

長鰭鮪(長鰭金槍魚)

Thunnus sp.

correct

correct

Tuna Longfin tuna with spicy sauce Albacore tuna

Fish Fish

KP259549.1 GU946662.1

98-99% Thunnus alalunga 98-99% Thunnus alalunga

Fish

KP259549.1

AC C

K2 X2 P2 F3 U1

TE D

W7

EP

X6 W1

RI PT

希靈魚

X1

I2

吞拿魚

N/A

Fish

KU955344.1

99%

Thunnus atlanticus

Blackfin tuna

黑鰭鮪(黑鰭金槍魚)

Thunnus sp.

correct

N/A

Z2 a

N/A

Tuna

Fish

KU955344.1

99%

Thunnus atlanticus

Blackfin tuna

黑鰭鮪(黑鰭金槍魚)

Thunnus sp.

N/A

correct

Mislabeling

0/4

0/4

Xiphias gladius

correct

correct

Swordfish sushi B3 劍魚

Swordfish

Fish

GU946671.1

99%

Xiphias gladius

Swordfish

劍旗魚(劍魚)

*processed

*processed

wrong piece given

*with sauce

ACCEPTED MANUSCRIPT

Tilapia sushi A1 立魚

B7

立魚

Swordfish belly Fatty swordfish

Fish Fish

GU946671.1 GU946671.1

99% 99%

Xiphias gladius Xiphias gladius

Swordfish Swordfish

劍旗魚(劍魚) 劍旗魚(劍魚)

Tilapia

Fish

GU477628.1

99%

Oreochromis niloticus

Nile tilapia

尼羅口孵非鯽

Snapper

Fish

GU477628.1

99%

Oreochromis niloticus

Nile tilapia

尼羅口孵非鯽

Sea eel

Fish

AB278890.1

99%

Anguilla japonica

Japanese eel

Z7 b

Eel tail

Fish

AB278890.1

99%

Anguilla japonica

Japanese eel

煙鯖魚

飛魚子軍艦

Not determined

correct

correct

Not determined

correct

wrong

Mislabeling

0/2

1/2 (50%)

Anguilla japonica

N/A

correct

日本鰻鱺

Anguilla japonica

N/A

correct

Mislabeling

N/A

0/2

*cooked and with sauce *cooked and with sauce

100%

Scomber scombrus

Atlantic mackerel

鯖(大西洋鯖)

Scomber scombrus

correct

correct

*cooked and marinated with vinegar

N/A

AB120717.1

99%

Scomber scombrus

Atlantic mackerel

鯖(大西洋鯖)

Scomber scombrus

correct

N/A

*cooked and marinated with vinegar

Mislabeling

0/2

0/1

Flying fish roe roll / "Crab roe" roll Z6 a N/A Crab roe Y1

correct correct 0/3

KJ128897.1

Flying fish roe

Other sushi Z1 炭燒鰹魚刺身 Katsuo Tataka 盒 A4 多春魚籽 Shishamo Roe

Sushi with unclear fish type

Fish

TE D

D5

correct correct 0/3

Mackerel in vinegar Fish

Roe

AB444863.1

99%

Cheilopogon antoncichi

Spotfin flyingfish

安東鬚唇飛魚

Cheilopogon sp.

N/A

wrong

Roe

KY067950.1

99%

Cheilopogon cyanopterus

Margined flyingfish

青翼鬚唇飛魚

Cheilopogon sp.

correct

correct

Mislabeling

0/1

1/2 (50%)

EP

Mackerel sushi B2 醋鯖魚

AC C

N/A

日本鰻鱺

M AN U

Eel sushi Z4 a N/A

Xiphias gladius Xiphias gladius Mislabeling

RI PT

劍魚腩 劍魚

SC

C4 F4

Fish

KM605252.1

99%

Katsuwonus pelamis

Skipjack tuna

鰹

Clustered to Katsuwonus

correct

correct

Roe

GU233808.1

99%

Mallotus villosus

Capelin

毛鱗魚

Mallotus villosus

correct

wrong

*cooked and with sauce

ACCEPTED MANUSCRIPT

澗白（壽）

Akirabeni

Fish

AB517558.1

99%

Seriola dumerili

Greater amberjack

杜氏鰤(高體鰤)

Seriola dumerili

wrong

wrong

no such English, Chinese or Japanese common name

L3

白玉豚壽司

Oilfish

Fish

KR231708.1

99%

Lepidocybium flavobrunneum

Escolar

鱗網帶鯖(異鱗蛇鯖)

correct

wrong

A2

紅魚

Frozen red fish slice Fish

EF446548.1

99%

Sebastes alutus

Pacific ocean perch

革平鮋

Lepidocybium flavobrunneum Sebastes sp.

wrong

wrong

Z8 b

N/A

N/A

AP002949.1

99%

Pagrus major

Red seabream

日本真鯛 (真赤鯛(真鯛))

Pagrus major

N/A

N/A

meaning correct in oilfish naming only by its red skin unlabeled piece in sushi set

Fish

M AN U TE D

From sushi mix set B

EP

From sushi mix set A

b

AC C

a

SC

Mislabeled samples are highlighted in grey.

RI PT

U2

ACCEPTED MANUSCRIPT Table 2. Taxonomic discrimination of fish sushi at species- or genus-level with 16S rRNA fragment by phylogenetic clustering Discriminatory level

Species

Species-level

Salmo salar (Fig. S1a), Oncorhynchus keta, Oncorhynchus

RI PT

kisutch, Oncorhynchus gorbuscha (Fig. S1a, Fig. S1b), Mallotus villosus (Fig. S4), Seriola dumerili, Seriola quinqueradiata (Fig. S5) Paralichthys olivaceus (Fig. S7), Xixiphia gladius (Fig. S9), Anguilla japonica (Fig. S11),

Scomber scombrus (Fig. S8), Lepidocybium flavobrunneum (Fig. S12), Pagrus major (Fig. S14)

Cheilopogon sp. (Fig. S2), Clupea sp. (Fig. S3), Thunnus sp.

SC

Genus-level

AC C

EP

TE D

M AN U

(Fig. S8), Sebastes sp. (Fig. S14), Atheresthes sp., Pseudopleuronectes sp. (Fig. S6)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights 93 pieces of sushi were collected in Hong Kong for 16S rRNA sequencing analysis. 16S rRNA sequence identified most fish in sushi products at genus or species level. Overall rate of mislabeling in English was 26.0% and that in Chinese was 17.8%.

AC C

EP

TE D

M AN U

SC

RI PT

English labels translated from Japanese had a high rate of mislabeling (40%).