Seafood consumption is highly demanding due to the important source of protein it contains, as well as being rich in omega-3 fatty acids. However, the adulteration of seafood is an alarming issue worldwide, including India. This study deals with edible crabs from seafood shops on the Odisha state coast in eastern India. The generated DNA barcode sequences successfully identified most of the studied brachyuran crab species by similarity search results in global databases. The species were also delimited by significant genetic divergence and Neighbour-Joining phylogeny. Additionally, the study detected the contamination of unknown organisms in the commercialized crab recipes from seafood shops. The DNA based species detection of brachyuran crab may be useful to resolve many ambiguities in species identification and monitoring of commercialized seafood concerning food safety.
Seafood is one of the ‘highly dealt lucrative commodities’ around the world (1). Due to the high consumption rate, importation and the globalization of the seafood industry, over 8000 tons of marine species (i.e. fishes, crabs, prawns, squids etc.) were captured by fishermen communities during 2010-2011 in India (2). However, in food safety concern of each commodity; food regulators are enforcing proper labeling of food products based on the composition and purity (3). The economically motivated food fraud is a risk that is gaining recognition and concern in recent days (4). The adulteration of seafood products is gradually increasing in various coastal regions throughout the globe and is posing a greater risk to consumer health (5,6).
The sea coast of the Odisha state lies in between 21.61N, 87.48E and 19.12N, 84.79E and is known as a popular tourist place in India. The total length of the coastline is about 480 kilometres, covering six districts. The region has a unique biodiversity composition, from wide spread mangroves, to many endemic and rare faunal elements (7). The coastal and offshore waters also form a rich abode of many crustacean resources (8). After marine fishes, the brachyuran crabs and prawns are some of the most highly consumed seafood, with a high demanding market value. Every year many people visit this place for recreation purposes and for seafood consumption. Unfortunately, the consumers are often unable to detect the cooked or untagged seafood recipes offered by the locals due to the missing of external morphology.
The survey of brachyuran crabs in Odisha state was started long back ago (7). A total of 140 species of brachyuran crabs belonging to 79 genera of 30 families has been recorded from a wide range of habitats in the Odisha coast (8,9). Earlier, the classifications of brachyuran crabs were described based on the shape, size, texture of carapace and buccal frame (9). Later on, the position of gonopore was charaterized as an important taxonomic character for accurate species identification (10). However, the study with gonopore is difficult for a non-taxonomist. The partial fragment (~650bp) of mitochondrial DNA (mtDNA), the Cytochrome C oxidase subunit I (mtCOI) gene, has been standardized to identify the brachyuran crabs (11). So far, very limited studies have been conducted for generating DNA barcode data from taxonomically identified brachyuran crabs in India (12). The molecular tools have also not been used to detect the seafood products being commercialized in the coastal regions of India. Therefore, this research aimed to determine the efficacy of mtCOI gene to identify the brachyuran crabs from a seafood shop on the Odisha coast. This baseline DNA data would enrich the global database and substantiate the identification of brachyuran crabs from India, as well as help to detect the commercial seafood fraud.
Materials and Methods
Sampling and laboratory analysis
A total of nine samples (six whole body brachyuran crabs and three amorphous fresh tissue samples processed for crab recipes) were collected from seafood shops in three different locations: Chandipur (21.44N 87.01E), Chandbali (20.79N 86.67E) and Gopalpur (19.23N 84.88E) in Odisha state (Figure 1). The whole body specimens were preliminary identified by available taxonomic keys (10,13). The muscle tissues were obtained from the chelate leg of whole specimens, preserved in 70% alcohol and deposited in the Crustacea Section of Zoological Survey of India, Kolkata. Further, the amorphous fresh tissue samples were directly collected in 500μl ATL buffer containing 50mM Tris-HCl (pH 8.0), 25mM EDTA (pH 8.0), and 150mM NaCl, for DNA analysis. The total genomic DNA was extracted by standard Phenol-chloroform extraction method with Proteinase K (200μg/ml) (14). The extracted DNA was checked by 1.5% Agarose gel electrophoresis using standard protocol. The published primer pair was used to amplify the partial mtCOI gene segment (15). The 25μl PCR reaction mixture contains 10 pmol of each primer, 10-20 ng of DNA template, 1x PCR buffer, 1.0-1.5 mM of MgCl2, 0.25 mM of each dNTPs, and 0.25 U of high-fidelity TaqDNA polymerase. The thermal profile for PCR was set as initial denaturation at 94oC for 2 minutes, followed by 30 cycles at 94oC for 45 seconds, 50oC for 45 seconds and 72oC for 1 minute, and subsequent storage at 4oC. The amplification was performed using a Veriti® Thermal Cycler. The PCR products were purified by using QIAquickR Gel extraction kit and cycle sequencing products were cleaned using standard BigDye X Terminator Purification Kit. The bidirectional sequencing was performed by the 48 capillary array, Applied Biosystems 3730 DNA Analyzer, in the in-house sequencing facilities of Zoological Survey of India, Kolkata.
Similarity search, genetic distance and phylogenetic analysis
The low quality regions were trimmed at both end and ambiguous bases greater than 2% were discarded, using a quality value of >40 for both bidirectional chromatograms reads. The nucleotide BLAST (BLASTn) program was used to further evaluate the sequences with no gaps, and the ORF finder to examine the complete alignment of protein coding genes without stop codons (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Finally the generated sequences were submitted in global database (GenBank) to acquire the specific accession numbers. Further, based on the BLASTn results, eight closely related sequences were acquired from the GenBank database. The generated and published database sequences were aligned using ClustalX software to form a combined dataset (16). The generated sequences were identified through the online identification system, in GenBank with ‘Highly similar sequences (megablast)’ and BOLD databases with ‘All Barcode Records on BOLD’ search engine. The genetic distances of the dataset sequences were analyzed through the Kimura 2 parameter (K2P) model in MEGA6, due to the transitional and transversional substitution rates. Further, to test the monophyletic clustering of the studied species, Neighbour-Joining (NJ) phylogeny was established by using MEGA6 with the K2P model and 1000 bootstrap replications (17).
Results and Discussion
The DNA barcode sequences detected five brachyuran crabs, Portunus pelagicus (family Portunidae), Tubuca rosea (family Ocypodidae), Neosarmatium asiaticum (family Sesarmidae), Galene bispinosa (family Xanthidae), and Matuta planipes (family Matutidae) with 98% to 100% similarity to the same species in both GenBank and BOLD database (Table 1). However, one sample (ZSI_CR11) showed 96% similarity with the Calappa lophos (Accession no. KX757761, generated from the southern coast of India) in GenBank. The C. lophos (family Calappidae) is a highly argued species widely distributed in the Indian Ocean to the western Pacific, including the Andaman Sea, Japan, Taiwan, and Australia (18). Due to the low identity, as depicted in the similarity search result, we further examined the genetic divergence of generated and publicly available database sequences of C. lophos. The incongruency in genetic divergences between the two published sequences of C. lophos and the generated sequence (high genetic divergence with AY579999: collected from Phuket, Pichai Fish Port, Thailand and low genetic divergence with KX757761: collected from the southern coast of India) depicts inconclusive identification of C. lophos in this study. This study remarks that the generation of more DNA barcode data of C. lophos from different geographical regions would further authenticate the understanding about the species.
|Voucher ID||Morphological identification||Accession No.||Highest similarity search in GenBank||Highest similarity search in BOLD|
|ZSI_CR1||Portunus pelagicus||MF043861||99||P. pelagicus||99.69||P. pelagicus|
|ZSI_CR2||Tubuca rosea||MF043862||98||U. rosea||98.08||T. rosea|
|ZSI_CR4||Neosarmatium asiaticum||MF043863||99||N. asiaticum||99.34||N. asiaticum|
|ZSI_CR7||Charybdis vadorum||MF043864||100||C. vadorum||100||C. vadorum|
|ZSI_CR8||Galene bispinosa||MF043865||100||G. bispinosa||99.85||G. bispinosa|
|ZSI_CR10||Tissue sample||MF043866||83||Octolasmis hawaiense||83.46||Balanus balanus|
|ZSI_CR11||Calappa cf. lophos[*]||MF043867||96||C. lophos||96.02||C. lophos|
|ZSI_CR12||Tissue sample||MF043868||84||Sacculina sp.||83.02||Sacculina sp.|
|ZSI_CR14||Matuta planipes||MF043869||98||M. planipes||99.5||M. planipes|
The N. asiaticum is distributed from Sri Lanka through the Andamans, ending in Indonesia and Taiwan (19). In this study, we detected the genetic data of N. asiaticum from the sample (ZSI_CR4) obtained from seafood shops. We assumed the occurrence of N. asiaticum from the same locality, since the fishermen community caught the marine fauna near the seafood shop location, and sold the same products to the seafood shops (personal observation). Hence, the study provides the genetic record of N. asiaticum in the coastal region of the Odisha state, and assumes a new distribution record of the species. With this preliminary observation, further rigorous taxonomic studies can be completed to confirm the distribution of N. asiaticum in Odisha. Further, the sequence generated from the amorphous fresh tissue sample (ZSI_CR7) shows 100% similarity with Charybdis vadorum (family Portunidae) in both NCBI and BOLD databases. The DNA data of two more tissue samples (ZSI_CR10 and ZSI_CR12) shows 83% to 84% highest similarity with the species (Octolasmis hawaiense, Balanus balanus, and Sacculina sp.) of class Maxillopoda in both GenBank and BOLD database, and thus the crab species identification remains unknown in this case. Nevertheless, this study suspected contamination of unknown organisms in the commercialized crab tissue samples, which are being processed for crab recipes. Therefore, this study suggests adoption of proper management to avoid the seafood contamination in the coastal region of India.
In conclusion, this study detected a total of six edible brachyuran crab species from the seafood shops. The intra-species K2P genetic divergence among the dataset ranged from 0% to 1.9%. The NJ tree resulted monophyletic clade of each studied species with 100 bootstrap supports. In this dataset, G. bispinosa and C. lophus show a close relationship with M. planipes and T. rosea respectively. Further, the C. vadorum show a close relationship with P. pelagicus and N. asiaticum, forming a distinct clade from all studied species (Figure 2). The generated sequences of the unknown organisms here associate as an out-group. Thus, the utility of DNA barcodes to detect seafood products in this case study revealed a number of implications pertaining to correct species identification and management efforts (20). The contributed DNA data in the global database further helps to strengthen the species identification library. Further, the tagging of DNA data offers proper regulatory compliance in the export of seafoods in international markets (21). Moreover, the technique is emerging as a reliable tool to assure all consumers concerning food authentication or food safety.
We thank the Director of ZSI, MoEF&CC for providing necessary permissions and facilities. We acknowledge the research funding from ZSI, MoEF&CC in-house project to the second author VK and DST-SERB National Post-Doctoral fellowship (F. No. PDF/2015/000302) to the third author SK. The funders had no role in study design, data collection and analysis or preparation of the manuscript.
1 Wong, E.H.K., Hanner, R.H. (2008). DNA barcoding detects market substitution in North American seafood. Food Research International, 41, 828-837. Search in Google Scholar
2 Kumar, S.T., Shivani, P. (2014). Marine Fisheries; Its Current Status, Sustainable Management and SocioEconomic Status of the Marine Fishers of Odisha, Through Indian Marine Policy: A Case Study. Research Journal of Animal, Veterinary and Fishery Sciences, 2, 10-19. Search in Google Scholar
3 Harris, D.J., Rosado, D., Xavier, R. (2016). DNA barcoding reveals extensive mislabeling in seafood sold in Portuguese supermarkets. Journal of Aquatic Food Product Technology, http://dx.doi.org/10.1080/10498850.2015.1067267. Search in Google Scholar
5 Carvalho, D.C., Palhares, R.M., Drummond, M.G., Frigo T.B. (2014). DNA barcoding identification of commercialized seafood in South Brazil: A governmental regulatory forensic program. Food Control. 10.1016/j.foodcont.2014.10.025. Search in Google Scholar
6 European Parliamentary Research Service. (2014). Fighting Food Fraud. http://www.europarl.europa.eu/RegData/bibliotheque/briefing/2014/130679/LDM_BRI(2014)130679_REV1_EN.pdf; accessed in 13/10/2014. Search in Google Scholar
7 Kemp, S. (1915). Fauna of Chilika Lake. Crustacea, Decapoda. Memoirs of Indian Museum, 5, 201- 325. Search in Google Scholar
8 Dev Roy, M.K., Nandi, N.C. (2009). Notes on the distribution of brachyuran crabs inestuarine ecosystems of east coasts of India. Journal of Natural History, 5, 7-11. Search in Google Scholar
9 Rao, D.V., Rath, S. (2014). Fauna of Brahmani-Baitarani Estuarine Complex, Odisha, Bay of Bengal w.s.r. to Ichthyofauna and Crustaceans. Estuarine Ecosystem Series. Records of the Zoological Survey of India, 7, 1-115. Search in Google Scholar
10 Ng, P.K.L., Liu, H.C., Wang, C.H. (1997). On the terrestrial crabs of the genus Neosarmatium (Crustacea: Decapoda: Brachyura: Grapsidae). Journal of the Taiwan Museum, 49, 145-159. Search in Google Scholar
11 Ahyong, S.T., O’Meally, D. (2004). Phylogeny of the Decapoda Reptantia: resolution using three molecular loci and morphology. Raffles Bulletin of Zoology, 52, 673-693. Search in Google Scholar
12 Vartak, V.R., Narasimmalu, R., Annam, P.K., Singh, D.P., Lakra, W.S. (2015). DNA barcoding detected improper labelling and supersession of crab food served by restaurants in India. Journal of the Science of Food and Agriculture, 95, 359-66. Search in Google Scholar
13 Alcock, A. (1900). Materials for carcinological fauna of India. No. 6. The brachyurian Catametopa, or Grapsoidea. Journal of the Asiatic Society of Bengal, 69, 279-456. Search in Google Scholar
14 Sambrook, J., Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Volume 1. Cold Spring Harbor Laboratory Press, Science - 2344. Search in Google Scholar
15 Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294-299. Search in Google Scholar
16 Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876-4882. Search in Google Scholar
17 Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 2725-2729. Search in Google Scholar
18 Lai, J.C.Y., Chan, W.K., Ng, P.K.L. (2006). Preliminary molecular and morphological study of the Calappa lophos species group (decapoda:brachyura:calappidae). Journal of Crustacean Biology, 26, 193-205. Search in Google Scholar
19 Ragionieri, L., Fratini, S., Schubart, C.D. (2012). Revision of the Neosarmatium meinertispecies complex (decapoda: brachyura: sesarmidae), with descriptions of three pseudocryptic indo-west pacific species. Raffles Bulletin of Zoology, 60, 71-87. Search in Google Scholar
20 Stern, D.B., Nallar, E.C., Rathod, J., Crandall, K.A. (2017). DNA Barcoding analysis of seafood accuracy in Washington, D.C. restaurants. PeerJ, 5, e3234. Search in Google Scholar
21 Rasmussen, R.S., Morrissey, M.T. (2008). DNA-Based Methods for the Identification of Commercial Fish and Seafood Species. Comprehensive Reviews in Food Science and Food Safety, 7, 280-295. Search in Google Scholar
© 2018 Shibananda Rath et al., published by De Gruyter
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