A molecular forensic method for identifying species composition of processed marine mammal meats

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Short report

A molecular forensic method for identifying species composition of

processed marine mammal meats

Chia-Hao Chang, M.Sc., Student

a,b

, Chiou-Ju Yao, Ph.D., Research Fellow

c

,

Hsin-Yi Yu, M.Sc., Student

d

, Yun-Chih Liao, Ph.D., Research Fellow

a

,

Nian-Hong Jang-Liaw, Ph.D., Research Fellow

e

, Chi-Li Tsai, Ph.D., Research Fellow

f

,

Kwang-Tsao Shao, Ph.D., Research Fellow

a,*

a_{Biodiversity Research Center, Academia Sinica, Taipei, Taiwan, ROC}

b_{Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC} c_{Department of Biology, National Museum of Natural Science, Taichung, Taiwan, ROC}

d_{Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan, ROC} e_{Animal Department, Taipei Zoo, Taipei, Taiwan, ROC}

f_{Endemic Species Research Institute, Nantou, Taiwan, ROC}

a r t i c l e i n f o

Article history:

Received 30 September 2013 Received in revised form 15 November 2013 Accepted 19 January 2014 Available online 25 January 2014 Keywords:

Meat Cetacea Phocidae

Cytochrome oxidase I Wildlife Conservation Act Illegal trade

a b s t r a c t

We used universal primers designed for the cytochrome oxidase I (CO I) sequence of the order Cetacea and the family Phocidae to prove that meat fritters sold in Taiwan contained meat from two seal, six cetacean, and one pig species. The sequence information for CO I obtained in this study was limited and population genetics data for the eight sampled marine mammalian species was insufﬁcient to deduce where these marine mammals were hunted. Regardless of the geographic origins of the marine mammal ﬂesh, sale and consumption of marine mammals in Taiwan violates the Wildlife Conservation Act. This study provides PCR primers that could enable government testing of suspect meats to curtail the illegal trade in marine mammal products.

1. Introduction

Cetacea are loveable marine creatures that draw public atten-tion to the issues of biodiversity and environmental conservaatten-tion. Animals of the Cetacea order have a long history as a versatile natural resource for humans. People have used their meat, an an-imal source of protein, for food, and their teeth and bone as ma-terials for traditional jewelry. Also, whale oil is still used as a fuel, an industrial lubricant, and a component of margarine. However, modern technologies mean cetacean tissue is no longer an indis-pensable ingredient in many products. To conserve marine biodi-versity, we should cease consuming cetaceans, particularly since many cetacean creatures are endangered. Currently, the sustainably managed whale watching business, rather than the traditional

whaling industry, offers the means to accomplish the goal of pre-serving these endangered species. There are 27 cetacean species recorded in Taiwandall of them are protected by the government of Taiwan.1

Traditionally, cetacean meat has provided a protein supplement for postpartum women and has been used as an ingredient in meat fritters, giving them a special_{flavor; they were especially popular in} Yunlin and Chiayi counties in Taiwan.2 In 1989, the Taiwan gov-ernment enacted the Wildlife Conservation Act, which protects all cetacean species in Taiwan from activities such as trading, hunting, and display. Despite this law, in January 2013, Taiwanese mass media reported that Cetacean meat fritters were being sold in Yunlin County, although the vendors claimed that they were made with seal meat imported from Canada.3It was difficult to verify the vendors’ assertions at the time of sale because the meat fritters are marketed as homemade, as a non mass produced item. Scientific appraisal of the animal species composing the meat fritters is ongoing.

* Corresponding author. Tel.: þ886 2 27899556; fax: þ886 2 27883463. E-mail address:[email protected](K.-T. Shao).

Contents lists available atScienceDirect

Journal of Forensic and Legal Medicine

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o ca t e / j fl m

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Molecular forensics employs genetic markers to identify the species or the speciﬁc individuals represented by a sample. The technique is widely applied to traditional medicinal materials, foods and animal products in order to prevent overexploitation of protected species and to seize smuggled material.4e10In this study, we propose a suitable set of primers for identifying cetacean meat with a barcoding technique, thereby facilitating forensic examina-tion of marine mammal products.

2. Materials and methods

2.1. Sample collection and DNA extraction

A total 20 meat fritters (Fig. 1) were collected from four different itinerant vendors in Yunlin County, Taiwan in 2013. We analyzed ﬁve samples from each vendor, as detailed inTable 1. Two pieces of meat were randomly selected from each meat fritter for testing. We extracted 40 DNA samples from the meat tissues using a Quick Gene DNA tissue Kit S (Fujiﬁlm, Tokyo, Japan).

2.2. Primer design, and polymerase chain reaction (PCR)

In order to design a pair of universal primers, we downloaded 62 cytochrome c oxidase subunit I (CO I) haplotypes from 46 cetacean and 16 Phocidae species (Table 2) from GenBank and identified conserved regions with MEGA 5 software.11_{The pair of} primers, SP-F (50-CHG CHC AYG CHT TYG TRA TA-30) and SP-R (50 -ARY ATD GTR ATN CCD GCY GC-30) were created and tested by PCR ampli_{fication of 16 Cetacean samples obtained from the} National Museum of Natural Science (NMNS), Taiwan. PCR am-plifications of the partial barcode region located at the CO I po-sition (366 bp) were performed with 100 ng template DNA, 12.5

m

mol of each speciﬁc primer, SP-F and SP-R, 12.5

m

L of Fast-RunTMAdvanced Taq Master Mix (ProTech, Taipei, Taiwan), and distilled water in aﬁnal volume of 25

m

L. Thermal cycling began with one cycle at 95 C for 4 min, followed by 35 cycles of denaturation consisting of sequential steps of 95C for 0.5 min, 45 C for 0.5 min, and 72 C for 0.5 min, ending with a single extension step at 72C for 5 min. We puriﬁed the PCR products using a PCR DNA Fragment Extraction Kit (Geneaid, Taipei, Taiwan). Approximately 50 ng of the puriﬁed PCR product pre-pared with the SP-F primer was sequenced with an ABI PRISM BigDye Sequencing Kit (PE Applied Biosystems, Foster City, CA, USA).

2.3. Data analysis

We identiﬁed the species of each ampliﬁed barcode haplotype using the Barcode of Life Database (BOLD) (http://www. boldsystems.org/) and its statistical tools.

3. Results and discussion

The universal primers, although not tested by PCR of the phocid samples, were validated by positive results when they successfully ampliﬁed the barcode region of the 16 cetacean specimens from the National Museum of Natural Science, Taiwan (Fig. 2).

The effectiveness of PCR amplification can be diminished or eliminated by food processing conditions, including physical stress, high temperature, pH, and exposure to enzymatic activity, because these may destroy the primary structure of DNA.12,13Fortunately, DNA extracted from the sterilized meat (121C for 15 min) qualified for enlarging a DNA segment to about 350 bp14; moreover, the accuracy of molecular identification based on the DNA barcode region sequence approaches 95% when the sequence size is 300 bp.15Since the length of the amplified DNA segments in this study was 366 bp, our molecular forensic identification is consid-ered authentic. The barcode region sequences were successfully obtained from the 40 DNA specimens; among these 40 sequences, we identified 17 different haplotypes (Table 1).

Species identiﬁcation with BOLD showed that the 17 haplotypes came from two seal species (Cystophora cristata and Phoca groen-landica), six cetacean species (Grampus griseus, Delphinus delphis, Kogia breviceps, Steno bredanensis, Tursiops truncatus, and Feresa attenuata), and one pig (Sus scrofa). The similarity values of all haplotypes evaluated by BOLD ranged from 100% to 98.62% (Table 1). The haplotypes HS1N5-2, HS1S3, HS2N3-1, HS3-1, and HS4-1 all had 100% similarity values, which fully supports that they are C. cristata, P. groenlandica, S. scrofa, C. cristata, and Tursiops truncates. The HS1-1, HS4N1-1, HS4N3-2, and HS4N5-1 haplotypes had 99.72% similarity values, and the HS1-2, HS2-1, and HS3N1-2 had 99.45% similarity values. The HS2-2 and HS4N4-1 both had 99.17% similarity values, and the others, HS2N2-2, HS2N4-2, and HS3N4-2, had similarity values between 99% and 98%.

The threshold for species delimitation in the barcode region is debatable, with 1%, 2%, or 3% being adopted by different re-searchers.16e18Given that the similarity values of the 16 cetacean samples from NMNS, with the exception of Kogia sima (NMNS12932), ranged from 100% to 98.9%, so we chose 2% as the threshold for species delimitation in this study. Since the genetic evidence suggests that the K. sima may contain two different spe-cies,19the low similarity value of the NMNS12932 may indicate that the NMNS12932 represents one K. sima species and the specimen recorded on the BOLD represents the other. When we used the 2% threshold for species delimitation, all haplotypes fully accom-plished molecular authentication.

A rapid and accurate test to identify cetacean meat would fundamentally improve efforts to minimize smuggling and illegal exploitation. Compared to the immune colloidal gold strip meth-odology,20this PCR-based technique is superior because it enables species identi_{ﬁcation and is performed with extracted DNA rather} than muscle tissue.5,21,22

The goal of identifying phocid and cetacean haplotypes was achieved by successfully amplifying the barcode segments with the designed primers. Based on the mammalian phylogeny,23 the Cetacea, the Phocidae, and the pig all belong to the Laurasiatheria, and the pig is phylogenetically closer to the Cetacea than the Pho-cidae is to the Cetacea; hence, it is reasonable that the universal primers designed from the conserved regions of the Cetacea and the Phocidae could also be applied to the pig. Moreover, these Fig. 1. Cross section of sampled meat fritters. It is suspected that the dark red meat

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proposed universal primers may also make ampliﬁcation of the barcode region of all the laurasiatherian organisms feasible.

To sum up, the barcoding results from the sampled meat fritters conclusively prove that both phocid and cetacean meat were sold on the Taiwanese market, thereby violating the Wildlife Conser-vation Act. Moreover, seals are exotic to Taiwan, and seal products cannot be legally imported into Taiwan according to Taiwan’s laws and regulations; furthermore, there is no record of imported seal products in the Bureau of Foreign Trade (http://cus93.trade.gov.tw/ FSCI). We consequently conjecture that the seal meat was trans-ported into Taiwan illegally. Three out of four of the vendors

sampled sold meat fritters containing cetaceanﬂesh. The barcode data suggest that the D. delphis meat from Vendor B came from at least three individuals, and the S. bredanensis meat from Vendor D came from at least two different individuals. These six sampled cetacean species are all native to Taiwan; nevertheless, it is not possible to use genetic information in the barcode sequences to prove whether or not these six cetacean species were hunted in Taiwan. Therefore, criminal trafﬁcking could also be contributing to the illegal cetacean meat trade.

Consumption of marine mammal ﬂesh violates the law and harms the human body.24e26We have prepared a pair of universal Table 1

Sources of meat fritters and 16 cetacean samples from the National Museum of Natural Science (NMNS), Taiwan, along with DNA barcoding results. Locality/museum Date of purchase Sample code/ voucher number Haplotype code/ scientiﬁc name BOLD result (similarity%) Common name Vendor A Taihsi, Yunlin

County

Feb. 2013 S1N1-1 HS1-2 Phoca groenlandica (99.45%) Harp seal

S1N1-2 HS1S3 Phoca groenlandica (100%) Harp seal

S1N2-1 HS1-1 Phoca groenlandica (99.72%) Harp seal S1N2-2 HS1-1 Phoca groenlandica (99.72%) Harp seal S1N3-1 HS1-1 Phoca groenlandica (99.72%) Harp seal S1N3-2 HS1-1 Phoca groenlandica (99.72%) Harp seal

S1N4-2 HS1-2 Phoca groenlandica (99.45%) Harp seal

S1N5-2 HS1N5-2 Cystophora cristata (100%) Hooded seal Vendor B Tungshih,

Yunlin County

Apr. 2013 S2N1-1 HS2-1 Grampus griseus (99.45%) Risso’s dophin S2N1-2 HS2-1 Grampus griseus (99.45%) Risso’s dophin

S2N2-1 HS2-2 Delphinus delphis (99.17%) Common dophin

S2N2-2 HS2N2-2 Delphinus delphis (98.62%) Common dophin

S2N3-1 HS2N3-1 Sus scrofa (100%) Pig

S2N3-2 HS2-1 Grampus griseus (99.45%) Risso’s dophin

S2N4-2 HS2N4-2 Delphinus delphis (98.9%) Common dophin

Vendor C Mialiao, Yunlin County

Apr. 2013 S3N1-1 HS3-1 Cystophora cristata (100%) Hooded seal S3N1-2 HS3N1-2 Phoca groenlandica (99.45%) Harp seal

S3N2-2 HS3-1 Cystophora cristata (100%) Hooded seal S3N3-1 HS3-1 Cystophora cristata (100%) Hooded seal

S3N4-2 HS3N4-2 Kogia breviceps (98.9%) Pygmy sperm whale

Vendor D Tungshih, Yunlin County

Apr. 2013 S4N1-1 HS4N1-1 Steno bredanensis (99.72%) Rough-toothed dolphin S4N1-2 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin S4N2-1 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin S4N2-2 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin S4N3-1 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin S4N3-2 HS4N3-2 Feresa attenuata (99.72%) Pygmy killer whale S4N4-1 HS4N4-1 Steno bredanensis (99.17%) Rough-toothed dolphin S4N4-2 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin

S4N5-1 HS4N5-1 Sus scrofa (99.72%) Pig

S4N5-2 HS4-1 Tursiops truncatus (100%) Common bottlenose dolphin NMNS NMNS16960 Balaenoptera acutorostrata Balaenoptera

acutorostrata (99.45%)

Common minke whale NMNST18057 Tursiops aduncus Tursiops aduncus (100%) Indo-Paciﬁc bottlenose dolphin TCSN-SC0901 Sousa chinenesis Sousa chinenesis (100%) Chinese white dolphin NMNS1321 Grampus griseus Grampus griseus (100%) Risso’s dolphin TCSN-OO9901 Orcinus orca Orcinus orca (100%) Killer whale

NMNS1320 Steno bredanensis Steno bredanensis (99.72%) Rough-toothed dolphin NMNS2375 Stenella coeroleualba Stenella coeroleualba (99.72%) Striped dolphin TCSN-PE9901 Pepnocephala electra Pepnocephala electra (99.72%) Melon-headed whale NMNS4401 Pseudorca crassidens Pseudorca crassidens (100%) False killer whale NMNS5240 Lagenodelphis hosei Lagenodelphis hosei (99.45%) Fraser’s dolphin NMNS14583 Feresa attenuate Feresa attenuate (99.72%) Pygmy killer whale NMNS12932 Kogia sima Kogia sima (91.94%) Dwarf sperm whale TCSN-NP9303 Neophocaena phocaenoides Neophocaena phocaenoides (98.9%) Finless porpoise NMNS1871 Physeter macrocephalus Physeter macrocephalus (100%) Sperm whale NMNS5400 Ziphius carvirostris Ziphius carvirostris (100%) Cuvier’s beaked whale TCSN-MD9602 Mesoplodon densirostris Mesoplodon densirostris (96.97%) Blainville’s beaked whale

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primers to amplify partial barcode sequences of phocid and ceta-cean DNA. Our analysis of meat fritters conﬁrmed that marine mammals were illegally captured in Taiwan and/or illegally im-ported. The results of this study provide primers that the govern-ment could use for testing aimed at identifying illegal trade in marine mammalﬂesh. We appeal to the authorities to expand ef-forts to stop the exploitation of these marine mammals and to enhance biodiversity conservation and protection of public health.

Ethical approval None. Funding

None declared. Conﬂict of interest

The authors report no con_{ﬂicts of interest. The authors alone are} responsible for the content and writing of the paper.

Acknowledgments

The authors feel obliged to Mr. Li, Ming-Chang and Ms. Chen, Jhen-Nien for their assistance in collecting meat fritters.

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Table 2

The GenBank accession numbers of 46 cetacean and phocid CO I sequences used to design the pair of universal pairs.

Taxon Scientiﬁc name GenBank accession no. Cetacea Balaenoptera borealis EU496284

Balaenoptera acutorostrata EU496285 Balaenoptera physalus EU496282 Balaenoptera edeni EU496283 Eschrichtius robustus EU496281 Megaptera novaeangliae EU496287 Eubalaena glacialis EU496286

Kogia breviceps EU496307

Kogia sima EU496308

Physeter macrocephalus EU496279 Mesoplodon bidens EU496312 Mesoplodon mirus EU496309 Mesoplodon carlhubbsi EU496310 Mesoplodon europaeus EU496313 Mesoplodon densirostris EU496311 Ziphius cavirostris EU496280 Delphinapterus leucas EU496288 Pseudoorca crassidens EU496319 Globicephala melas EU496303 Globicephala macrorhynchus EU496299

Grampus griseus EU496295

Peponocephala electra EU496291 Feresa attenuata EU496289

Orcinus orca EU496323

Steno bredanensis EU496375 Sotaliaﬂuviatilis EU496374

Sousa chinensis EU496345

Stenella frontalis EU496350 Stenella clymene EU496346 Stenella coeruleoalba EU496341 Stenella longirostris EU496331 Stenella attenuate EU496339 Lagenodelphis hosei EU496355 Tursiops aduncus EU496330 Tursiops truncates EU496324 Delphinus capensis EU496371 Delphinus delphis EU496360 Lagenorhynchus albirostris EU496357 Lagenorhynchus acutus EU496356 Pontoporia blainvillei EU496358 Inia geoffrensis EU496359 Phocoena phocoena EU496315 Neophocaena phocaenoides EU496316

Phocoena sinus EU496314

Phocoena spinipinnis EU496317 Phocoenoides dalli EU496318 Phocidae Erignathus barbatus AY377143 Cystophora cristata AY377144

Phoca vitulina NC_001325

Phoca largha AY377147

Phoca groenlandica AY377145

Pusa hispida AY377146

Pusa caspica NC_008431

Halichoerus grypus NC_001602 Monachus monachus AY377142 Monachus schauinslandi AY377141 Mirounga angustirostris AY377138 Mirounga leonine AY377140 Lobodon carcinophagus AY377130 Ommatophoca rossii AY377132 Leptonychotes weddellii AY377136 Hydrurga leptonyx AY377134

Fig. 2. Positive results of 16 cetacean species ampliﬁed by PCR with the designed primers, SP-F and SP-R. The scientiﬁc name with the specimen voucher number in parentheses of the PCR product in each loading lane is as follows. L1: Balaenoptera acutorostrata (NMNS16960), L2: Tursiops aduncus (NMNST18057), L3: Sousa chinenesis (TCSN-SC0901), L4: Grampus griseus (NMNS1321), L5: Orcinus orca (TCSN-OO9901), L6: Steno bredanensis (NMNS1320), L7: Stenella coeroleualba (NMNS2375), L8: Pepnoce-phala electra (TCSN-PE9901), L9: Pseudorca crassidens (NMNS4401), L10: Lagenodelphis hosei (NMNS5240), L11: Feresa attenuate (NMNS14583), L12: Kogia sima (NMNS12932), L13: Neophocaena phocaenoides (TCSN-NP9303), L14: Physeter macrocephalus (NMNS1871), L15: Ziphius carvirostris (NMNS5400), and L16: Mesoplodon densirostris (TCSN-MD9602).

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