Molecular genetics provides a powerful tool for conservation of species protected by international

regulations or threatened by overexploitation. The present communication is the first report from

India on the application of molecular tools for the accurate identification of a stranded whale in

putrefied condition as it was impossible to identify the species status using conventional taxonomy

and the carcass of an unknown animal devoid of its head and tail, collected from a fish market.

Partial sequences of mtDNA control region and cytochrome b gene of the whale were generated and

tested with BLAST search and DNA surveillance for molecular identification. It was identified as

Bryde's whale (Balaenoptera edeni). Partial sequence of mtDNA cytochrome b gene of the unknown

fish from the market was generated, tested with BLAST search and was identified as sword fish

Xiphias gladius

Afsal, V V

Anoop, B

Jayasankar, P

Peter, Reynold

Rajagopalan, M

CMFRI Digital Repository

339Indian J. Fish., 54(3) : 339-343, 2007Species of a whale and an unknown fish sample identifiedusing molecular taxonomyP. JAYASANKAR*, B. ANOOP, REYNOLD PETER, V. V. AFSAL ANDM. RAJAGOPALANCentral Marine Fisheries Research Institute, Cochin - 682 018, India*E. mail :  jayasankarp@vsnl.comABSTRACTMolecular genetics provides a powerful tool for conservation of species protected by internationalregulations or threatened by overexploitation. The present communication is the first report fromIndia on the application of molecular tools for the accurate identification of a stranded whale inputrefied condition as it was impossible to identify the species status using conventional taxonomyand the carcass of an unknown animal devoid of its head and tail, collected from a fish market.Partial sequences of mtDNA control region and cytochrome b gene of the whale were generated andtested with BLAST search and DNA surveillance for molecular identification. It was identified asBryde's whale (Balaenoptera edeni). Partial sequence of mtDNA cytochrome b gene of the unknownfish from the market was generated, tested with BLAST search and was identified as sword fishXiphias gladius.Accurate identification of species isfundamental to conservation efforts ofbiodiversity. Conventional morphology-basedtaxonomy often fails to offer unambiguousidentification of groups showing littleevolutionary differentiation, cryptic membersof species complexes, members of closelyrelated species that can only be identified at aparticular life stage, fry, fingerlings or juvenilesof cultured species, inter-species hybrids andalso in the verification of illegal fishing as wellas marketing of endangered species. Moleculartechniques have become a major tool forsystematic biologists at the species level andabove.DNA sequence analysis is a powerful toolfor conservation - identifying the source ofsamples thought to be derived from threatenedor endangered species. The technique could beeffectively used in the forensic identificationof commercial products and verification oftrade records (Baker et al., 1996). The DNA-based approach would help the conservationistto identify the species even from a small pieceof tissue sample, such as skin from the marketedproduct. Besides marine mammals, molecularbased taxonomic identification has greatapplication in validation of marketed species,such as sea turtles, sharks and sturgeons (Dizonet al., 2000).The present paper describes two cases ofspecies identification carried out by moleculartaxonomic approach. One was a baleen whaleof about 12m in total length stranded on thebeach at Kundugal near Mandapam (Gulf ofMannar), Tamil Nadu on 8th August 2006. Itwas in a decayed condition making itimpossible to identify the species status usingmorphological characters (Fig 1). The skintissue in a portion of the caudal fluke was almostintact and about 3 g of skin tissue was sampledfrom this region and preserved in 70% ethanol.Total genomic DNA was extracted using thestandard phenol-chloroform methodNote340(Sambrook et al., 1989) with slightmodifications as follows: about 25 mg of skintissue was finely chopped and placed inextraction buffer (containing 0.1M NaCl,10 mM Tris HCl, 1mM EDTA), with 10% SDSand digested by  proteinase K (20 µl) at 65ºCfor 3 h. This was followed by the routine phenol,chloroform:isoamyl alcohol extractions and theextracted DNA was resuspended in TE andstored at - 20ºC until further use.PCR was performed on PTC100 (MJResearch) thermocycler in a total volume of25 µl containing 10-100 ng of extractedgenomic DNA template, 10 mM of Tris-HCl(pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.01%gelatin, 240 µM each of dATP, dTTP, dCTP anddGTP, 1 U of Taq DNA polymerase and10-25 pM each of forward and reverse primers.Table 1 shows the particulars of the primersused in this study to amplify mtDNA controlregion and cytochrome b gene. The temperatureprofile for the amplifications was an initialdenaturation at 94ºC for 2 min followed by 35cycles of 94ºC for 1 min, 54ºC - 57ºC for 2 minand 72ºC for 1 min and a final extension at 72ºCfor 7 min. Quality of the PCR products waschecked on 1.5% agarose gel and the productswere purified using the KT 62 Genei quick PCRpurification kit.Cycle sequencing reaction was performedwith the purified PCR product based on ABIAmpliTaq FS dye terminator cycle sequencingchemistry on a ABI 3100 PE automatedcapillary sequencer in presence of the forwardprimer, reaction buffer and the fluorescentlylabeled dye terminators for the required numberof cycles at specific temperature. The sequenceswere first edited using Bio Edit ver 7.0.5.3(Hall, 1999), aligned using the computersoftware Clustal W multiple alignments(Thompson et al., 1994) with gap opening 10,gap extension 0.2 and bootstrap 1000 andcorrected manually. The primer and otherambiguous sequences were deleted. Nucleotidesequences of cyt b gene were translated toamino acid sequences using the software PrimerPremier ver 5.00.Molecular identification of the whalesample was done in two steps: initially sequencesimilarity search of the edited user sequencewas done in BLAST (Basic Local AlignmentSearch Tool) contained in a comprehensivedatabase, GenBank (www.ncbi.nlm.nih.gov)TABLE 1: Particulars of the mtDNA primers used in the studyLocus Primer sequence Annealing PCR Sourcetemp (0C) productsize(bp)Cytochrome b GLUDG-L (5’-TGACTTGAARAACCAYCGTTG-3’) 54 432 Palumbi (1996)CB2-H (5’-CCCTCAGAATGATATTTGTCCTCA-3’)Control region M13-Dlp1.5-L  (5’- 54 527 Dalebout et al.TGTAAAACGGCCAGTTCACCCAAAGCTGRARTT (1998)CTA-3’)Dlp5-H (5’-CCATCGWGATGTCTTATTTAAGRGGAA-3’)Fig. 1. Beach-stranded baleen whale (later identifiedas Bryde's whale) in a highly putrefiedconditionP. Jayasankar et al.341(Benson et al., 2007). Once it was confirmedthat the tissue sample was from a cetacean(dolphin, porpoise or whale), the speciesidentity was searched with the database of DNAsurveillance (www.cebl. auckland.ac.nz: 9000)(Baker et al., 2003, Ross et al., 2003). Thesequences, after their confirmation, weresubmitted in GenBank using a standalone multi-platform submission program called Sequin(www.ncbi.nlm.nih.gov/Sequin/index.html).Partial sequences of rapidly evolvingmtDNA D-loop (control region) andcytochrome b gene were generated and testedto identify the whale sample. The BLASTsearch showed 99-100% sequence similaritywith Bryde's whale (Balaenoptera edeni). TheDNA surveillance search also unambiguouslyshowed the species of the whale as Bryde'swhale (Figs. 2 and 3). The sequences weredeposited in the GenBank under accessionFig. 2. Neighbour joining tree of mtDNA control region partial sequence of Balaenoptera edeni (M6, GenBankaccession # EF057443) based on reference sequences in DNA surveillance. The numbers on the treebranches indicate bootstrap valuesSpecies of a whale and an unknown fish sample identified using molecular taxonomyFig. 3. Neighbour joining tree of mtDNA cytochrome b gene partial sequence of Balaenoptera edeni (M6,GenBank accession # EF057444) based on reference sequences in DNA surveillance. The numberson the tree branches indicate bootstrap values0.05 Expected Substitutions per SiteseiseiseiBryde’s (common)Bryde’s (common)Bryde’s (common)Bryde’s (Kochi)M6 EF057443Bryde’s (Kochi)Bryde’s (Kochi)Bryde’s (omurai)Bryde’s (omurai)spermsperm100100 pygmy rightpygmy right1001005191959999 99968975Bryde’s (Kochi)M6 EF057444seisperm0.01 Expected Substitutions per SiteseiBryde’s (common)Bryde’s (common)Bryde’s (Solomon Is.)blue (pigmy?)blueblueAntarctic minkeAntarctic minkeAntarctic minkeAntarctic minkeNorth Pacific minkeNorth Pacific minkeNorth Pacific minkeNorth Atlantic minkedwarf minkegraygraygraygraygrayfinfinfinhumpbackhumpback1001008610010055100659999905983896010074999310052567484342numbers EF057443 (mtDNA control region)and EF057444 (mtDNA cytochrome b gene).The second case was a headless andtailless animal carcass observed on 14th October2006 in the Ernakulam fish market.  The carcasslooked much like a dolphin with thick blackskin. A piece of skin tissue was collected fromits dorsal fin and proceeded with the protocolsdescribed above for molecular-basedidentification of the species. BLAST search ofthe sequence (Fig. 4) from the GenBank(www.ncbi.nlm.nih.gov) produced anunexpected result. The mtDNA cytochrome bgene sequence of the source 'animal' showed99% sequence similarity with that of sword fish,Xiphias gladius.These two case studies proved the utilityof molecular techniques to identify the speciesof stranded marine mammal accurately even inthe absence of whole intact animal to examinethe morphological characters as well as in theidentification of unknown samples. It is evenmore significant that it could be achieved froma small piece of skin tissue of the dead animal.P. Jayasankar et al.AcknowledgmentsThe authors are grateful to the Ministry ofEarth Sciences for financial assistance to carryout this work. Thanks are also due to theDirector, CMFRI, Cochin for the facilitiesprovided and to Dr. R. Paul Raj, PrincipalScientist, for critically going through themanuscript.ReferencesBaker, C. S., F. Cipiano and S. R. Palumbi 1996.Molecular genetic identification of whale anddolphin products from commercial markets inKorea and Japan. Mol. Ecol., 5: 671-685.Baker, C. S., M. L. Dalebout, S. Lavery and H. A.Ross 2003. www.DNA-surveillance: Appliedmolecular taxonomy for species conservationand discovery. Trends in Ecol. Evol., 18:271-272.Benson, D. A., I. K. Mizrachi, D. J. Lipman, J. Ostelland D. L. Wheeler 2007. GenBank, Nuc. Acids.Res., 35: D21–D25.Fig. 4. Cytochrome b partial sequence of the fish sample (later identified as sword fish, Xiphias gladius)10 20 30 40 50 60 7080 90 100 110 120 130 140150 160 170 180 190 200 210220 230 240 250 260 270 280290 300 310 320 330 340 350360 370 380 390 400 410 420430 440 450GGATCTGCTG CAAGATATCA TTCTGATGGG CTCCGACTAA ACCCACCCCC TCCTAAAAAT CGCAAACGAC 70GCACTAGTGG ATCTCCCAAC TCCCTCCAAC ATTTCAGTCT GATGAAACTT CGGCTCCCTC CTCGGCCTCT 140GTTTAGCTGC CCAAGTCCTC ACAGGCCTGT TTCTCGCTAT ACACTATACG TCAGACATCG CGACGGCCTT 210TACATCCGTA GCACATATCT GCCGAGATGT AAATTATGGA TGACTCATCC GAAACATGCA CGCCAACGGC 280GCATCCTTCT TCTTCATCTG CATTTACCTC CACATTGGCC GAGGTCTTTA CTATGGCTCC TATCTATACA 350AAGAAACATG GAATATTGGA GTTGTTCTCC TTCTCCTAGT AATGATGACC GCTTTCGTAG GTTATGTATT 420ACCCTGAGGA CAAATTCCAA ATTCTGAGGG 450343Date of Receipt : 20-06-07Date of Acceptance : 31-08-07Species of a whale and an unknown fish sample identified using molecular taxonomyDalebout, M. L., A. V. Helden, K. V. Waerebeek andC. S. Baker 1998. Molecular identification ofsouthern hemisphere beaked whales (Cetacea:Zhiphiidae). Mol. Ecol., 7: 687-694.Dizon, A., S. Baker, F. Cipriano, G. Lento, P. Palsbolland R. Reeves 2000. Molecular geneticidentification of whales, dolphins andporpoises: proceedings of a workshop on theforensic use of molecular techniques to identifywildlife products in the marketplace. NOAATech. Memo., 286: 1-51.Hall, T. A. 1999. BioEdit- a user-friendly biologicalsequence alignment editor and analysisprogram for Windows 95/98/NT. Nuc. Acids.Symp. Ser., 41: 95-98.Palumbi, S. R., 1996. Nucleic Acids II: ThePolymerase Chain Reaction. In: MolecularSystematics, p. 205-247, D. M. Hillis, C. Moritzand B. K. Mable (Eds.). Sinauer Associates,MA.Ross, H. A., G. M. Lento, M. L. Dalebout, M. Goode,P. McLaren, A. G. Rodrigo, S. Lavaery andC. S. Baker 2003. DNA surveillance: web-based molecular identification of whales,dolphins and porpoises. J. Heredity, 94:111-114.Sambrook, E., F. Fritsch and T. Maniatis 1989.Molecular Cloning, Cold Spring Harbour,Newyork: Cold Spring Harbour press.Thompson, J. D., D. G. Higgins and T. J. Gibson1994 CLUSTAL W: Improving the sensitivityof progressive multiple sequence alignmentthrough sequence weighting, position specificgap penalties and weight matrix choice. Nuc.Acids. Res., 22(22): 4673-80.

Species of a whale and an unknown fish sample identified

using molecular taxonomy

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Species of a whale and an unknown fish sample identified using molecular taxonomy

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