IDENTIFICATION OF ANISAKIS SIMPLEX USING DNA BARCODING
Introduction
DNA barcoding provides fast and reliable identification of all animal species, using approximately 655 base pair long portion of mitochondrial cytochrome c oxidase subunit I (COI) gene. Barcoding could be used to uncover violations of labeling requirements in processed or raw seafood (Keskin and Atar 2011, 2012) and identification of parasites sampled from commercially important aquatic species. This study presents, first barcoding data regarding molecular identification of Anisakis simplex (Rudolphi, 1809) found in Merlangius marlangus (Linnaeus, 1758) specimens sampled from Black Sea.
Material and methods
Whitings, sampled from Black Sea (Sinop, Turkey) were examined. A total of three specimens yielded 10 (3,4,3) nematodes. All the nematode specimens were subjected to DNA extraction using Qiagen DNeasy Blood and Tissue Kit. Mitochondrial COI gene was amplified using the primer pair HCO (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') and LCO (5'-GGTCAACAAATCATAAAGATATTGG-3') (Folmer et al 2005) for nematode specimens; FishF1 (5ˈ-TCAACCAACCACAAAGACATTGGCAC-3ˈ) and FishR1 (5ˈ-TAGACTTCTGGGTGGCCAAAGAATCA-3ˈ) (Ward et al. 2005) for fish samples. PCR reactions were set according to Keskin and Atar (2011, 2012). All fish and nematode sequences were conducted on an ABI 377 automated sequencer. Reference COI sequences of representative taxa were obtained from NCBI GenBank and BOLD databases. Phylogenetic analyses were conducted on MEGA 5.1.
Results
Whiting specimens were identified as M. merlangius as expected. Nematodes were identified as A. simplex (L4 stage). Average nucleotide composition for A. simplex specimens were calculated as A=19.3%, C=13.9%, G=21.8% and T=45.0%. Intraspecific Kimura 2-parameter mean genetic distance was 0.004 (0.4%). BLAST comparisons resulted with maximum identity percentage of 99 for all specimens. Similarity percentage values calculated by BOLD database were between 98.61%-99.02%. DNA barcoding tree constructed using BOLD Taxon ID Tree (Fig. 1) clustered our A. simplex specimens under the node including A. pegreffii (Campana-Rouget & Biocca, 1955) and A. simplex specimens, under the branch containing only A. simplex species.
Discussion and Conclusion
Current results presented in this study would be used as a molecular reference to identify Anisakis simplex specimens in species level, which makes it possible to identify this nematode even at different life stages. Barcoding could be a useful molecular tool for discovering intermediate hosts, elucidating parasite life cycles (Siddall et al. 2012) and identification at species level.
References
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