STANDARD OPERATING PROCEDURE

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MD/SP/24(93)

ISBN 978-983-9114-60-7 SEAFDEC/MFRDMD/SP/24

STANDARD OPERATING PROCEDURE

for

Tissue Sample Collection and Preservation of Sharks and Rays

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STANDARD OPERATING PROCEDURE

for

Tissue Sample Collection and Preservation of Sharks and Rays

MARINEFISHERYRESOURCESDEVELOPMENTANDMANAGEMENTDEPARTMENT(MFRDMD) SOUTHEASTASIANFIS

TAMANPERIKANANCHEND

•MENTCENTER(SEAFDEQ ALATERENGGANU,MALAYSIA HERIESDEVELOP

)ERING,21080KU^

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PerpustakaanNegaraMalaysia DataPengkatalogan-dalam-Penerbitan

STANDARDOPERATINGPROCEDUREforTissueSampleCollectionand PreservationofSharksandRays/byMARINEFISHERY

RESOURCESDEVELOPMENTANDMANAGEMENTDEPARTMENT (SEAFDEC/MFRDMD)

Bibliography:pages 13 ISBN978-983-9114-60-7

I. Sharks—Identification.2.Rays(Fishes)—Identification.

3.Education—Methodology.

597.3 Preparedby:

NoorulAzlianaJamaludin WaliidahMohdArshaad MasayaKatoh

AdamLukePugas

This publicationshouldbecitedasfollows:

Noorul-Azliana J., Wahidah MA.，Katoh M. and Luke P.A. (2014). Standard Operating ProcedureforTissue SampleCollectionandPreservationofSharksandRays.

SEAFDEC/MFRDMD/SP/24.

Printedby

T^crcdn^/Ub YAYASANISLAM

TERENGGANUSON.OHD.

GONG8ADAK.21300KUALATERENGGANU,T Tel:09-6688611.6&66652.6668601faks:09-S66TERENGGANU

•0611/6660063

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This StandardOperatingProcedures (SOP)servesasaguidelineandreferenceforsharksand rays tissue collection/sampling. Tissue collected is analysed using molecular biology technique alternative identification method. Currently DNA barcoding research sharksisprogressing intheworld.ItisexpectedthatDNAdataonsomeelasmobranchspecies in the region

performed on sharks and rays caught in Malaysia waters. Genetic research on some ofthe unsequenced species is also conducted. High biodiversity in the region makes this project sophisticatedandmorethan 170speciesofelasmobrancheshavebeenrecorded.Theexpected outputs forthe project include the biological information of sharks and rays in the region, which can be used for development and management of sharks and rays in the region.

as an on

available. Compilation ofthe available data and species identification

are are

2.0 OBJECTIVE

Study onbiologyofmajorelasmobranch(sharksandrays) species,providebasicknowledge forconservation andenhancementofsharkandraypopulationsin theregion. Morerecently,

regionallevelthepressuretolistcommerciallyimportantandvaluablemarinespecies CITES is growing. Identification ofelasmobranch species is fundamental ofbiological data collection.Expertiseonidentificationandbiologicaldatacollectionon sharksandraysinthe regionneedtobestrengthened.

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3.0SAMPLINGATPORTS 3.1Point of concern

a. Samplinglocation.Speciesidentificationby authorizedtaxonomist

b. Freshsample givesbetterDNAextraction.Samplescollectedmust befromonlyproperly board of the fishing vessel. This is to ensure freshness of the preserved catch

fishsampled.

on

c. Need to maintain the freshness of fish until the tissue is sampled and preserved.

Sampledfishes atthesampling site should bekeptin acontainerwith iceor dry ice to maintainthefreshness ofthesamples untiltissue collectionandpreservationactivities isdone.Thisparticularly importantin thecasewhen tissue collectionand preservation activitiesistobecarriedoutnotonlyinsitu(atthesamplingsite)butalsolateraftertaken backtolaboratory.(Referto4.3).(Notethat,oncepackedofcrashedice/dryice mustbe useduntiltissuepreservationisdone).

d. Possible cross-contamination when fish are sampled from mix-species container.

Ensuresampledfishareproperly wipedcleanof slimaround sample sitebeforetissue sampling.

3.2Materialsand toolsforsamplingatport

Figure 1:Materialsandtoolsused forsamplingatport

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Table1: Listofmaterialsand toolsforsamplingatport

Thisissuitablefortransportationofsamplefromsampling porttothelaboratory.Itssizedependingonthesample.

Coolingbox 1

Eachsamplemustbeattachedaproperidentificationlabel (AppendixI).

2 DataForm 1

3 Ice orDryIce

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This is oneofthe important items forgeneticsample collection. Sample amount should bepreparedfor the samplecollection.

木Notinthepicture

3.3Procedureforsamplingat port

Samples collectedand speciesidentification at landingsite.Include tagforeachsample.

1.

Fillform 1 (Appendix 1)

Putthesampleinto icebox tomaintainfreshness ofthesamples.

2.

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3. Thefishsamplesshouldbemaintainedcovered with crashice oruse ofdry ice inthe ice box untilthenextstepfortissuepreservation.Tissue preservationwillbedoneatlaboratory.

At laboratory, fish samples should be keptin freezer preferably at - 20°C until tissue preservationprocedureiscarried out.

*Proceed to 4.3 if the tissue decided to be preservedatthe sametime.

4.

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4.0TISSUESAMPLECOLLECTIONANDPRESERVATION

4.1 Pointofconcern

1. Needto maintain thefreshness ofthesample.Pelvic finshouldbetaken immediately after thesample fishwas taken outfrom the storage.The remaining ice/water must bewipedoutfromthesamplingarea.

2. Avoidcontaminationofthesample. Forcepsandscissors mustbe washedwithclean waterandethanol andbumtosterilizeeverytimebeforeuse.

3. Samplestorage temperatureisno longeran issue.The vialscontainingtissuesamplein buffer(ethanol) canbestoredatroomtemperature. Oncepreservedin ethanolsamples bestored formanyyears.Ethanolshouldbe checkedperiodically forevaporation.

Therefore,storagein fridgeorfreezerwillreduceethanolevaporation.

can

4.Tissueshouldbefullypreserved.Eachtissuesampleshouldbeplacedinindividual vials, approximately20mgoftissuefor 1.5 ml ofethanol.Nomore thanlA tissueto3A ethanol byvolume,overloadingthevials causesthetissuetobepoorlypreserved.

5. Sample without proper labelis problematic. Vials should be labelled with dissolvingethanolresistantmarkerorprintedlabels toavoidlossoflabel.

a non-

4.2Materials fortissuecollectionand preservation

Figure2: Listofmaterialsandtoolsfortissuecollection

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Table2:Listofmaterialsandtoolsfortissuecollection DESCRIPTION NAME

1. Setofforcepsandscissors Usetocuttissuesamplesfromfishbody.

Useforwashforcepsandscissors.

2. Wash bottlefilledwith ethanol(95%)

Useforrinseforcepsandscissors.

3. Washbottlefilled with clearwater

4. Burner oralcohollampor Useforsterilizingforceps andscissors.

lighter

Forplacingspecimenduringtissuecollection.

5. Tray

Inwhichtissuesamplesarepreservedwith buffer contained95 % ethanol.

6. Vialsfilledwith preservation buffer

Towipeoutthewaterandanyorganicsfromforcepsand scissors.

7. Tissuepaper

Towearduring samplingprocess.

8. Disposable gloves

Tolabelsamples.

9. Permanentmarker

Toweightthespecimens.

10.Weight Scale

To thespecimens.

11.MeasuringTape measure

Torecordtheinformation.

12.Form1 (fromport)

4.3Procedurefortissuecollectionandpreservation

1. Record allinformationofthe specimeninto Form 1.

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2 Labelthevialswithformatgiven.

(Species/Year/SampleNumber).:

Thevialshouldbelabelledwithspecies(e.g CcircharhinusbrevipinnaasC. brevipinna), date(year),samplingsite andvialnumber(as thevialnumberinForm2).

Example:

Species :C.brevipinna Year: 2013= 13 Samplingsite: Kuantan Samplenumber : 01 Code: Cb/13/KTN/01

Proceedtostep 3forsharksandstep4 forray.

3.Measuringandspecimenlabel forsharks

3a.Takepicture ofthe specimenwithscaleand tag.Proceedtostep5

4-Measuring andspecimenlabelforrays

4a.Takepictureofbothsurfacesofthesped with scaleandtag.Proceed tostep6.

imen

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5.Weightforshark

5a. Weigh thespecimenandrecordinForm 1.

5b. Measuretotallengthofthespecimen and recordinForm 1.

5c. Wipe ihesamplefishwith tissuepaper.

Proceedtonumber7.

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6.Weightand measurement

6a. Weighthespecimenand recordinForm 1.

6b. Measuretotallength/disclength/discwidth ofthespecimen andrecordinForm 1.

6c. Wipethesamplefishwithtissuepaper.

Proceedtonumber7.

7. Wash forcepand scissors withcleanwater andthenwipewithtissuepaper.

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! 8. Washforcep andscissorswith95%ethanol.

9. Burn scissorsand forcep for sterilization.

Note: Never touchthe edgesofsterilized tools.

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10. Cutapproximately30mgofpelvicfinofthe fish. Please avoid any contamination.

Proceedtonumber 12forthesharkspecies.

11. Cutapproximately30mgofpelvicfinofthe fish.Pleaseavoidanycontamination.

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12. Immediately,byusingforcep,placethecut tissueintoavial thatcontainedpreservation buffer.

Note: Always handle the tissue using sterilizedtools toavoidcontamination.

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13. Screwthevialcapthevialtightlyandplace inasafecontainer.

14. Sterilize the scissors andforceps before takingtissuesamplefromthenextspecimen:

Repeatsteps 1 to 14foreach sample.

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References

AbuTalib,A.,NoomlAzliana,J. &Wahidah,M.A. (2013).StandardOperatingProcedure forTissueSampleCollectionandPreservation.SEAEDEC/MFRDMD/SP/21.

Steinke,D.& Hanner,R.(2011).TheFISH-BOLCollaborators’Protocol.Mitochondrial DNA,22(S1): 10-14.

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APPENDIXI FORM1

TissueCollectionForm

SoutheastAsianFisheriesDevelopmentCenter

MarineFisheryResourcesDevelopmentandManagementDepartment TISSUECOLLECTIONFORMFORSHARKSANDRAYS Collector⑻

Samplingdate Samplingmethod Samplingsite Longitude/Latitude Commonname Scientificname Identifier

Weight Kg

TotalLength(TL) Cm

DiscLength(DL) Cm

DiscWidth(DW) Cm

Sex Male/Female/Unidentified Lifestage Juvenile/Adult/Unidentified Remarks

Forlaboratoryuse only:

木木

1/2/3/4/5 Sampleno.

Tissuecollection date

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APPENDIXH FlowChartforTissueSampleCollection Procedure

START

(Fish samplingatfishlandingport/site) SAMPLECOLLECTED&

SPECIESIDENTIFICATION

FILLUPFORM 1

PUTSAMPLESIN ICEBOX

(Tissuesamplecuttingand preservation) SENDTOLAB

OR

KEEP IN REFRIGERATOR

-20 °C RECORDALLTHE

INFORMATIONINFORM 1

TAKE PICTURE OF EACH SPECIMEN BOTH SURFACE (FOR RAY ONLY) WITH SCALE & TAG

WEIGHT, MEASURE EACH OF FISH AND RECORD INTO FORM 1

WASH FORCEP WITH CLEAR WATER WIPE FISH WITH

TISSUE PAPER WASH FORCEP WITH

95% ETHANOL

CUTTISSUEOF

BURNFORCEP FISH

PLACETISSUEINLABELLEDVIAL WITHFORCEP

SCREWVIAL CAP

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ISBN Vfl-邗3-mM-bO-?

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STANDARD OPERATING PROCEDURE

STANDARD OPERATING PROCEDURE

for

Tissue Sample Collection and Preservation of Sharks and Rays

W、.越

STANDARD OPERATING PROCEDURE

for

Tissue Sample Collection and Preservation of Sharks and Rays

Contents

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