Title Identification of dugong (Dugong dugon) tissues usingisozymes

Author(s) Kittiwattanawong, Kongkiat; Adulyanukosol, Kanjana;Buakaew, Pantarak Na

Citation Proceedings on the 4th SEASTAR2000 Workshop (2003): 77-80

Issue Date 2003

URL http://hdl.handle.net/2433/44137

Right

Type Conference Paper

Textversion publisher

Kyoto University

77

Identi鮎ationor血gong（上九JgO〝g血go乃）

tissues using isozymes

KoNGKIATX止m槻m触AWONG，KANJANAADUImOSOandPANTARAKNABuABnEW

邦〟たgf〟8rよ乃eβわわg血JCe〃おrf，0．動肌抑げ肋血相部恍 励血公転吻払り＠如励Ⅶ丑α棚

Aβ5TRACT

Twotissuespecimens，SuSPeCtedofbeingdugongweretestedbyanalyzngisozymes・Thefirsttissue SPeCimenwascorlectedfrom Ban PakIok，Phuketon26－hofOctober，2000andthesecond onewas COllectedfromafreshmarketinPhukettownon29thofOctober，2000・Thesuspectedtissueswere COmParedwithknowntissuesoffivedugongs（Dugongdugon），threecows（Bostaurus），threepigs （Susscrofa）・threechickens（Gallusdomesticus）andonefinless

．

glucose－6－Phosphateisomera＄e，lactatedehydrogenase，isocitratedehydrogenase，malate， mannose－6－Phosphateisomerase and phosphoglucomutase．Usingthe zymograms ofthese SeVenenZymeS，thefirsttissuespecimenwasidentifiedasdugongtissue，Whilethesecondtissue SPeCimenwasnot・Duetoincompatibilityofproteinpatterns，WeWereunabletoass■gnthesecond tissuespecimentoanyofthecomparedorganisms・An e】ectrophoreticanalysiso＝sozymeshas PrOVentObeaneffectivetoolforrecognitionofdugongtissueandshowspotentialforidentificationof Otherconserved organismsthatarepoached．

mODtJCTION

InTbiland，dugong（かugmgdugon）isoneoftheprotected

SeaanimalsbytheFisheriesAct1947andunderCITES

in1983・HtlntingandpossessionofdugonglnCludingltS

remainsareillegal（Adulyanukosol，2001）・Inthepast，

dugongscontributedtothedietofvillagersalongboth

COaStlinesoftheAndamanSeaandtheGulfofThailand．

However，thechanCeOfcatchingdugonglSVerylowbecause

Ofthepresentsmal1numbers・Althoughdugongshave

notbeenhunted，theyarecaughtaf［erbecomingentangled

inflShinggearespeciallygillnets（Boonprakobetal・，

1983；ChantrapornsylandAdulyanukosol，1994；

Adulyanukosol1995and1999）・

Mostoftbed11gOng’smusclesystemsresemble

Closelythoseofmanatees，eXCePttheshouldermuscles

thatarequitedifferent（DomIling1977inNishiwakland

Marsh，1985）．Altho11ghfreshmeatofdugongandthe

appearanceofbothtextureandpink－reddishcolorare

VerySimilartopork，thedugong’smeathaslesserfat

thanpork（Adulyan11kosol，明叫址血か坤椚相加）・

Furthermore，pOrkseemstodecomposeeasierthan

dugongrsneshandgenerallyporkissmelllerthandugong・

Howeverwithoutskln，itisqllitedifficulttodistinguish

thetwospecies・Soonaf（erthelocalvi11agersflndadead

d11gOng，SOmetimestheyillegallysellthemeatonthe

blackmarketoralternativelydistrib11temeatinsidethe

Vulage・

RecentlylnlateOctober2000，WereCeivedtwo

SamPlesof11nknowntiss11eS，Whichweresuspectedtobe

dugongslmeat・Thispapereval11ateSelectrophoresisof

isozymesasatoolforidentiflCationofdugongsamples・

MRIALSANDMETIIODS

rんrJ正†lJで．†

Thetwotissues，SuSPeCtedofbeingdugong，WereCOllected

fromBanPaklokandafreshmarket，Phuketprovinceon

2（油and29thofOctobeT，2000，reSpeCtively．mesetwo

tiss11eSWereSuSpeCtedasdugongmaterialbeca11SeOfthe

pinkrreddishcolor⊃Whichisthenormalcolorofdugong

m11SCle，The tissuesweresenttotheelectrophoresis

laboratory atPMBCinfreshcondltion・ThesllSpeCted

tlSSueSWereCOmparedwith銭veknownspeciestissues

Samples，i．e．鎖vedugongs，tbreecows（伽∫加r〟∫），three

pigs（Susscr（痴），threechickens（Gallusdomesticus），

andonefinlessporpoise（楠pphocaenaphocaenoides）

（Tもblel）・

創細物血椚血 TheanalysISWaSCarriedouton7r8thofNovember2001．

Atotalvolumeof30－60？lofl％polyvlnylpyrrolidone

（PVP）wasaddedtothetissuesamplestogetherwith

Sma11amotmtofcleanedsandbeforehomogenizing．Af（er

Centrifugingat4，105gfor5minutes，thesupematantS

Wereabsorbedontopaperwicks，Whichthenwereinserted

intostarchgels．Starchgelswerepreparedwith12％starch

7β

（SigmaS－4501）jntris｛itratebufEerpH7・0（Benzie，1993）・

Theproteinswereseparatedat600voltsand80mAfor

3hotlrS．StainlngWaSdonewithsevenenzymestainlng

recipesiLe■，dihydrolipoamidedehydrogenase（DDHor

diaphorase DIA，ECl．8．1．4），gl11COSe－Phosphate

isomerase（GPr，EC5．3Ll■9），1actatedehydrogenase

（LDH，ECl．1．1．27），isocitratedehydrogenase（IDH， NADPECl．1．1．42），malatedehydrogenase（MDH，EC l．1．1．37），mannOSe－6－phosphateisomerase（MPl，EC 5，3．1，8），Phosphoglucomutase（PGM，EC5L4・2・2）・The

stainlngreCIPeSWeredescribedbyHarris＆opkinson （1976）andManchenko（1994）・

Tablel・CollectlngSlteSandconditionoftheknown－SpeCiestissuesusedasreferencesforidentificationofsuspected tissues．FieldnumbersareidentiflCationcodesbrspeCimenskeptatPh11ketMarineBiologicalCenter・ Type：M＝≡muSCle，L＝1iver

Species Field Na. Type Collecting place Tissue candition

Decomposed，9－mOnth frozen

Semifresh，8－mOnth frozen

Semifresh，56－mOnth frozen

Semifresh，33－mOnth frozen

Fresh，Z（；－mOnth frozen

Fresh，1－W6ek frozen

Fresh，トweek frozen

Fresh，13－mOnth frozen

Fresll，nOt frozen

FresIl，nOtfrozen

Fresll，nOt frozen

M ToloyaiIsland，Trang

M LaemYong LalⅥ，Tra¶g

M KamIsland，Ranong

M WeallIsland，T＝ang

M Port of P壬Illket，Phuket

M paklok，Plluket

M Fresh market，Phuket

M Phangnga

M，L fresllmarket，Phuket

M，L Fresh market，Phuk（）t

M，L Fresh market，Phuket

か〃gO乃g血g‘フ乃（5） Du－119

Du－120

Du－048

Du－074

Du－084

Suspected tissue 1

Suspectedtissue

〃e呼ゐ∂Cde乃βクゐoc〟g〃Ogde∫（1）FINP166

β∂＝αⅣr〟∫（3）

∫〟∫∫Crq匝（3）

G〟JJ〟∫dome∫Jfc昆∫（3）

み椚（将川肌加fビワrg如ぬ〝

AzymOgramisdefinedasastrlPOrbandofelectrophoretic mediumshowiJlgthepatternofenzymesorisoenzymes aftertheirseparationbyelectrophoresis（Harrisand Hopkinson，1976）．Bandseparationismainlyd11etOthe netcharge，Sizeandshapeofprotein，Aproteinwith negativechargewi11runtOtheanodewhileproteinswith positivechargeswillruntOthecathode．nehigherthe Chargeaproteincontains，thefasteritcanrun．Incontrast， thebiggerthemolecularsizeofaprotein，theslowerit CanmOVealonganelectric銭eld■Azymogramderived fromeachenzyme－SPeCificstainlngmethodcaTIPOSSeSS morethanonelocusorsystem．Withinaloc11S，eaCh individualpossessesonetoseveralbandsdependingon Whetheritisheterozygoteversushomozygoteaswe11as Onformsofproteinse．g．monomere，dimereandtrimere （RichardsonetaL，1986）．ThesebandsarepheT10typesof allelesorgenes・However，nOteVerybandiscountedas anal1ele・Bandsofallelesateachlocuscanbedesignated n11menCallywiththefastestanode－mlgratingbanddenoted lll”，thesecondfastest▼▼2”，andsoon・ThetlSSueSPeClmenS

WereaSSlgnedasordifferentiatedfromdugongtlSS11eS byvisualcomparisonofthebands■locationswiththoseof OtherorganlSnRAsageneralru1e，ZymOgramSOfconspeciBc Organismstendtosharethesamelocations，Whilethe OneSOfdifFerentspeCiestendtolocatedistinctly（Hartl andClark，1989）・However，ithastobeemphasizedthat idendcalmigratlOnratedoesnotnecessari1yindlCateidentical aminoacidcompositionoftheproteins・

RESUIJTS

Zymogramsofseverl－eIIZymaticstainsareshownas Fig11rel・Bandsofsuspectedandd11gOngtissueswere developedwellforallenzymeSeXCeptIDH・Mostenzymes exhibitedbothanodicandcathodicmlgratlngbands exceptGPIwhichpossessedonlyproteinbandswith positivechargesLReferringtoMDH，DDH，LDII，MPI， andPGM，thefirstsuspectedtissue（Ul）wasassigned asdugongtissuewhereasthesecondone（U2）couldnot beasslgnedtoanyofthecomparedorganisms・

MDH：This enzymewas oneofthebestenzymesto differen血tedugongtissues丘omothercomparedorganlSmS・

Twozoneswereobserved，PrObablyrepresentlngtWO loci・7nefirstloc11S（anodicmigrating，1inel－6）hadsix

allelesandthesecondone（cathodicmigrating，1ine7－10）

hadfouralleles．FortheflrStlocus，bandofUllocatedat the6thlinewascommonwiththeonesofdugongtlSSueS Whileitdifferedfromothercomparedorganisms・U2was notcompatiblewithanyofcomparedorganisms．I）3did notshowanybands■Thesecondlocusshowedpalebands butwas sti11possibletoscore．Atthislocus，UIwas Iocatedonthesamelineasdugongtissues（1ine7），While difEerentiatedfronlOtherorganisms．

DDH：Tむozoneswereobserved，PrObablyrepresenting twoloci・However，therewereonlyheterozygotesin hens■musclesinthesecondloci（cathodicmigrating，1ine

乃

ー∴－一一 －l－ l

、－＿キ こ＿＿ ‾‾‾‾‾‾ ・

⊥＿ ：・

－＝〟

喜】㌣川棚＜～…

’－． －▲．．．－

OI－－－－丁●

；－ ‥ ニ i≡享；≡三≡

＝雷二子≡＝＝：二＝ 〟■ト

ダgg．了．gJec什呼んoノーeggCC8〝甲即f∫0／7∫げ仙0エ‘′一点〃0－V〃甲eC∫∽e〟∫r乙け〟〃d【佗）わ玩〃W〝平eCfme〃∫げ5－血go〃g∽〟∫CJe∫ 「伽g（〉〃g血g叫βJ＝刀旭－074エリ＝丑肝銅旦一刀＝伽JJpノか4＝刀厄一J2βフか∫＝か昆一J20ノ，ノー♪乃Je∫∫pOワOf∫e′m∫CJe 仲呼力ocβe〃叩毎c〟g〃∂fdeち叩けco？∫伽∫加叫CJ→3ノ，jザg∫侍〟∫∫Cr朝夕J‾刃α〟dj‾Cんざ血邦∫rGβ伽血me∫正c“∫∫ ・ 、 ＝ ミーー ・ 、、・ － ・・－・

ゐorizo〃rβJmⅣたf〃わg加ee花山dfc（Ife∫腕e∫fd畑乃gJf乃e．撒ビガg〟re∫0乃腕eJ申pβ〃eJ∫ゐ∂WOrよgf乃d／グ椚Ogr（‡椚ちW烏iJeo〃肋e

rよgJ－りフβ乃eαre砂椚Ogr‘‡∽f〃∫e岬reJ〟血柁∫・

6－8）．Inthefirstlocus（anodicmigrating，1inel－5），Ul tissuesbutnotinlivertissues・ WaSlocatedinthesameline（1ine2）withtheother dugongs（D4andD5）■Dlhadverypaleband，WhileD2 GPI＝Onezone

．

whichwerelocatedsimi1artocowtissues（Clr3）．In exhibitedheterozygotesexceptthelivertissues，Which chickensandpigs，thebandsonlydevelopedinmuscle werehomozygoteLTheknowndugongtlSSueS，aSWellas

βO

Ul，had3alleleslocatedatlinel，2and3．Thesedugong allelesresembledtheonesofcows．However，theywere Signi丘cantlydifferentfromallelesofchickensandpigsL U2hadtwoal1eles．Thefirsta11elewascIosetoline2， whilethesecondonewasbetweenline4and5．

IDH：Two zones were observed，agaln mOStlikely representingtwoloci（anodicmigratinglocus，1inelr3 andcathodicmigratinglocus，1ine4－5）・Noactivitieswere Obtainedfromsuspectedtissuesorfromdugongtissues・

LDH：Twozoneswereobserved，agalnmOStlikely representingtwol∝1（anodlCmigratlngloM，lJnel－7and Cathodicmigratinglocus，llJle7LlO）Inlocusl，heterozygotic allelesofUlresembledtheonesofd11gOngtissues（1ine2 and4）anddi批rentlatedfromotherorganlSmS・hlocus2， dugongtlSSueSaSWe11asUIsharedthesameaueleswith theonesofcows，andpigs（1ine7）．U2appearedonlyin 10CuS2andsharedthesameposition（1ine9）withchicken tissues．

MI）Ⅰ：Twozones weTe Observed，agaln mOStlikely representingtwolocl（anodlCmigratlngl∝出，11nel－7and Cathodicmigratnglα：瓜，1ine8－10）．hloml，UIwasonthe SamelmeasdugongtlSSueS（line7）andcouldbedistinguished fromothercomparedorgaJlismsaswellasU2・Inlocus2， thebandswerepaleandlocatedclosetoeachother・nis locuswasnotemployed鮫）rthedetermination・

PGM＝rIWozoneswere observed，agaln mOStlikely representingtwolocl（anOdlCmlgratingloc張，11nel－7and Cathodicmigratinglocus，line8－10）・Thelocusl，U2 revealed abandlocatedonthesameline astheonesof

COmpareddugoIlgtlSSueS（血e5）andcoddbedlStlngtllShed 丘omothercomparedorganismsincludingU2．Inlocus2，the bandswerepaleandnotusedfordeterminatlOnS，

CONCLUSIONS

Tsozymeelectrophoresishasbeenproventobeaneffective toolindetectingthesuspecteddugongtissue．Ourstudy SuCCeSSfu11yemployedflVeOutOfsevenenzymesstainmg recIPeStOaSSlgnthesuspectedtissueasdugongtissueand todifferentiatei抽omothercomparedorganismsi．e．丘nless POrPOISe，COWS，Chickens，andpigs・AmongflVeenZymeS， MDHrevealedthebestresultinbothbandintensity（easy todetect）andseparatingpotential（goodseparatlOnh）m OthercomparedorganlSmS）．However，inmostcases，the authorssuggestempIoyingal1nveenzymes簸）raCCurate detection．

Vlriationofbandintensltyreflectstheactivlty Ofenzymescontainedintissues，thefreshertheoriginal SamPle，thehlgherintensity・ThedugongtlSSueSuSedinthlS Studyvarledintheirirutialfreslmess（freshtodecomposed） andalsotermSOfdurationkeptina－200Cfreezer（8－56 months）LThest11dydemonstratedthatfresh－COllected dugongtissuefrozenfor24monthsaswellassemi－fresh－

collectedsamples丘ozenforupto33months＞areSu伍cient forisozymeelechphoresis・Longerpreservadonofsemi－＆esh tissuemaynotglVe11Sefu1results，Whilesamplesstored StOnngat－20oCfor56monthsshowednoactlVltylnmOSt enzymes・ThedecoPOSeddugongdssue（Du－119）proved dmostuselessforlSOZymeanalysIS・

ACⅨNOⅥrIJEDGEMENTS

Wearegratefultovi11agersofBanPaklokandMr・Chatchai Sarikhapan，StaffofPM】〕Cforprovidingthesamples fromBanPaklokandfreshmarket，reSpeCtively・Mr・Niyom Lohakam，ChiefofPhuketProvincialFisheryOfEice，and Mr・ThongchaiPathanakul，headoftheconservatlOnOf FisheryResourcesSection，thePhuketProvincialFishery Office，areaCknowledgedfortheirco－OPerationindugong researchandconservationinPh11ketprovince・Wealso appreciatetheedltingassistanceofVibekeSimonsenat A肌血旧thiversity，BabaraBrcwnatNewCastlethiversity， aswellasEllenHines，YvetteAIva，andFrankCiprianoat SanFrancis00StateUniverslty．

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