STANDAii - Operatiii Proce關疆

STANDAii

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O ^peratiii P ^roce 關疆

for Collection and Preservation ofDNfl Tissue Samples

SWEDEN

ISHlRIli Di^llLOPMlNfGlNTlR

ST ASU

S ^tandard O ^perating P ^rocedure for Collection and Preservation

of DNA Tissue Samples

Prepared by

Marine Fishery Resources Development and Management Department (MFRDMD)

Funded by:

SEAFDEC-Sweden Project

SWEDEN

SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER

Prepared by:

Wahidah Mohd Arshaad Raja Bidin Raja Hassan Noorul Azliana Jamaludin Abdul Razak Latun Mazalina Ali Mohammad Faisal Md. Saleh Annie Nunis Billy Mahyam Mohd Isa Adam Luke Pugas

SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER

MARINEFISHERIESRESOURCESDEVELOPMENT ANDMANAGEMENT DEPARTMENT(MFRDMD) FisheriesGardenChendering 21080KualaTerengganu, Malaysia TEL:+609 6175940,+609 617 1543,+609617 7867 FAX:+6096175136,+6096174042 EMAIL:mfrdmd@seafdec.org.my WEBSITE: www.seafdec.org.my

TABLEOFCONTENTS

1. INTRODUCTION

2. OBJECTIVEOFTHESOP

3. TARGETSPECIES...

4. IDENTIFIED SAMPLING SITES ²

5. SAMPLINGAT PORTS/LANDINGSITES... ... 4 5.1 Pointsofconcern...

5.2 Materialsandtoolsrequired fortissuesamplecollection...

5.3 Procedurefortissuecuttingandpreservation...

6. TISSUESAMPLECOLLECTIONAND PRESERVATIONPROCEDURES 6.1 Pointsofconcern...

6.2 Materialsandtoolsrequired fortissuesamplecollection...

6.3 Procedurefortissuecuttingandpreservation...

7. TRANSPORTATIONOFVIALSTOSEAFDEC/MFRDMD...

4 4 5

7 8 12 12 REFERENCES

LISTOFTABLES

Table 1:Identifiedsamplingsitesbycountry...

Table2:Listofmaterialsandtoolsforsamplingatport/samplingsites Table3:Listofmaterialsandtoolsfortissuecollection...

3 5 8 LISTOFFIGURES

Figure 1: TunaspeciesintheSoutheastAsianwaters...

Figure2: Identifiedsamplingsites...

Figure3: Materialsandtoolsusedforsamplingatport/samplingsites Figure4: Materialsandtoolsfortissuecollection...

.2 4

LISTOFAPPENDICES APPENDIXI: Form 1...

APPENDIXII:Form2...

APPENDIXIII:FlowChart

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INTRODUCTION

This Standard Operating Procedure (SOP) serves as a guideline and main reference for those involved intissuesamplecollectionatidentified samplingsites(Table 1) andtissue preservation in the field or at laboratory. Collected and preserved samples from the respectivecountriesaretobesenttoSEAFDEC/MFRDMD inMalaysiaforanalysis.

2. OBJECTIVEOFTHESOP

The main objectives ofthis SOP are to ensure thatall collected tissues are prepared and preserved according to the standard methods and procedures. The steps outlined in the SOP will ensure that sufficient high quality DNA could be obtained from the sampled tissues. High quality DNA is required to produce reliable and comparable data for stock/population identificationintheSoutheastAsianregion.

3. TARGETSPECIES

The keytogenus Thunnusofany taxonomic bookwould serveas a good reference. The page on “Tuna Identification Sheet” poster published from Project Information Collection ofHighly Migratory Species in Southeast Asian Waters: Tuna (2008-2012) isgivenforquickreference(Figure 1).

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TUNA IDENTIFICATION SHEET

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4. IDENTIFIED SAMPLING SITES

Sampleofthe samplingsites asshown inTable 1 andFigure2.

Figure2: Mapshowingthedistributionofthesamplingsites in theSoutheastAsian Region

2

Table 1: Sampling sites bycountry,sampling sites’ code,and number ofsamplesto becollected forgeneticstockstudyin theSoutheastAsian Waters

Country &

Sampling Site Code

No. of Sample Sampling Site/s

Country

Andaman Sea Sub-Region

> Indonesia INBA 50

INBW MYKP MMYN THRG 1. Banda Aceh

2. Belawan 3. Kuala Perl is 4. Yangon 5. Ranong

50 50

> Malaysia

> Myanmar

> Thailand

50 50 South China Sea and Gulf

of Thailand Sub-Region

> Brunei

> Cambodia

> Indonesia

> Malaysia

6. Muara Port 7. Sihanokville 8. Pemangkat 9. Tok Bali 10. Kuantan 11. Miri

12. Kota Kinabalu 13. Masinloc (Zambales) 14. Puerto Princesa (Palawan) 15. General Santos City 16. Trat

17. Songkla 18. Nghe An 19. Danang 20. Vung Tau 21. Kien Giang (GoT)

50 BRMP

CBSV rNPT MYTB MYKN MYMR MYKK PHMC PHPP PHGS THTR THSK VTNA VTDG VTVT - VTKG

50 50 50 50 50 50

> Philippines 50

50 50

> Thailand 50

> 50

> Viet Nam 50

50 50 50 Outgroup

> Indonesia

> Malaysia

> Philippines

INPN MYTU PHZA

50 22. Pekalongan

23. Tawau 24. Zamboaga

50 50

3

5. SAMPLING ATPORTS/LANDINGSITES 5.1 Points ofconcern

Eish samples must be collected from the landing sites listed in Table 1.

Information on where the fish are caught is crucial to ensure that the samples representthefishingareaas intended.

a.

b. Fresh sample gives betterDNA extraction. Samples collectedmustbe from only properlypreserved catch onboard fishing vessels. This is to ensure thefreshness ofthe fish sampled. Information on fishing location (latitude and longitude) and catchinggeartypemustbeobtainedandrecorded.

.c. Avoidotherthantargetedtunaspeciesinthe50sampledtissues.

Note: Country is required (ifpossible) to take photo for every fish sampled and sendthephotosto Laboratory. Photosofthe samplesmustbetakentogetherwith theIdentificationCodeNo.

d. Need to maintain the freshness offish until the tissue is sampled and preserved.

Sampled fishes at the sampling site should be kept in a containerwith ice or dry ice to maintain the freshness of the samples prior to tissue collection and preservation. This is particularly important in the casewhen tissue collection and preservation activities cannotbecarried out insitu (atthe sampling site) (Referto 5.2).

Sampledspawnerfish givesbetterindication inpopulationstudy.

Spawningindividualswillpreferablybecollected. Wherepossiblebiologicaldata for each sample such as weight, standard length, sex, and gonad development stage(Appendix IV) shouldberecordedduringsamplecollection.

e.

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

Ensuresampled fishareproperlywiped cleanofslimebeforetissuesampling.

5.2 Materialsand tools required fortissuesamplecollection

Figure3: Materialsand toolsused forsamplingatport/samplingsites 4

Table2: Listofmaterials and tools forsamplingatport/samplingsites DESCRIPTION NAME

This is used for packaging the sample, the size is dependingonthefishsizetobecollected

Plasticbag *

2 Container

(optional)

This is fortransportationofsamplesfromport/sampling site tothe laboratory; itssize dependsonthe numberof samples

3 Disposablegloves Tobeused forhandlingfishduring samplingprocessto minimizecontamination

4 DataForm 1 Each sample (in aplastic bag) must beattached with a properidentification label (DataForm 1 as inAppendix I)

5 Crushediceor Dryice *

This is important to maintain the freshness ofthe fish for genetic sample collection; ample amount should be prepared forthesamplecollection

5.3 Procedurefortissuecutting andpreservation

1. Fillupthe informationinForm 1

2. Writethereferencenumbertwice inForm 1

、\ 、

5

3. Cutoneofthenumbers inForm 1

4. Placeitinaplasticbagcontainingthesampled fish.

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5. Transfertheplasticbagcontainingsamples into container,thencoverthesampleswithcrushed iceorplacedryiceincontainer

6. Thefishsamplesshouldbe keptinthecontainer untilthenextstepfortissuecollectionand preservation,wheretissuepreservationcouldbe doneeitheratthelandingsite{insitu)orafter thesamplesarebroughtbacktolaboratory

*Pleaseproceedto6.0 iftissueisdecidedto be preservedinsitu(atthesamelandingsite)

7. Atthelaboratory,fishsamplesshouldbekept ina freezerpreferablyat-20°Cuntil tissue collectionandpreservationprocedure carriedout

are

6

6. TISSUESAMPLECOLLECTIONAND PRESERVATION PROCEDURES 6.1 Pointsofconcern

Need to maintain the freshness of the sample. Fin clip tissue should be taken immediately after the sample fish

ice/watermustbewiped awayfromthesamplingarea(theseconddorsalfin).

a.

taken out from the storage. The remaining are

b. Avoid contamination ofthe sample. Surgical gloves should be worn at all times during tissue sampling. Forceps and scissors must be washed with clean water andethanol everytimebefore use.

Avoid mixing ofsamples. The vials should be labeled clearly according to the format (Year/Country&SamplingSiteCode/SampleNumber), e.g. MALAYSIA, Tok Bali: 15/MYTB/01, THAILAND, Songkhla: 15/THSK/01, MYANMAR, Yangon: 15/MNYN/01. PleaserefertoTable 1 forcountryand samplingsitescode.

c.

d. Sample storage temperature is no longer an issue. The vials containing tissue sample, in buffer (ethanol) can be stored at room temperature. Once preserved in ethanol, samples can be stored for many years. Ethanol should be checked periodically for any evaporation. Therefore, storage in fridge

evaporation.

freezer will reduce ethanol or

Tissue should be fully preserved. Each tissue sample should be placed in individual vials, approximately 20 mg (1 cm2) oftissue in 1.5 ml ofethanol and ensure thattissuesampleisfullysubmerged.

e.

g. Sample without proper label is problematic. Vials should be labelled with a non­

dissolving ethanol resistant marker or printed labels or pencil to avoid possible lossoflabels.

6.2 Materialsandtools requiredfortissuesamplecollection

Figure4: Materials andtools fortissuecollection

Table3:Listofmaterialsandtoolsfor tissuecollection

NAME DESCRIPTION

Setofforcepsandscissors Forcuttingtissuesamplesfrom fish Forwashingforcepsandscissors Washedbottlefilledwith

ethanol(95%)*

2.

Forrinsingforcepsandscissors 3 Washedbottle filledwith

cleanwater

Forplacingspecimen duringtissuecollection 4. Tray*

Forpreservingtissuesampleswith95%non- denaturedethanol*

Vials filledwith preservationbuffer 5.

Forwipingawaywaterandanyorganicsfrom forcepsandscissors

6. Tissuepaper*

Tobeworn duringsamplingprocess Disposablegloves

7.

For labeling the vials containing samples and filling upforms

8. Permanentmarkerandpen

9 Weighingbalance* Forweighingsamples

Information on all sampled tissues must be filled in this form (information from Form I will also be copiedintoFormII)

10. DataForm2^ (AppendixII)

For measuring the standard length of the fish sampled

11. Measuringtape

6.3 Procedurefortissue cuttingandpreservation

1 Transferinformationaboutthesamplesfrom Form 1 intoForm2

(Note: Form 1 containsinformationon

fish/fishessampledatthesametimeandplace, while,Form 2containsinformationforthe50 sampledfishes)

8

2 Labelthevialswith theformatgiven

(Year/Country&SamplingSiteCode/SampleNumber):

Example:

Year: 2015= 15

CountryCode:Malaysia=MY SamplingSiteCode: TokBali=TB Sample number:01 (Ref. Form2) Code: 15/MYTB/01

3 Fillupeachvialwith95%non-denatured ethanol(approximately 1.5 ml)

4 Wipethesamplefishwithtissuepaper

5 Weigheach ofthesamplefish

6 Measuretheforklengthofthesamplefish

9

7 Capturefishphotowiththevial’sreference numberanddate

a

^{8 Fillupthe}informationonweightandfork

length(andifpossiblesexand gonadstage) (SEAFDEC/MFRDMD,2015)into Form2

9 Washforcepsandscissorswithcleanwaterand then95% non-denaturedethanolevery time beforeuse

10 Wipetheforcepsandscissorswith tissuepaper everytimeafterwashing

11 Cutapproximately30 mg(1 cm2/1.5cm x0.5 cm)finclipfromfirstdorsal finsofthesample fishwithsterilizedscissors

Note: Wipefinwithethanolbeforecutting

10

12 Immediately,usingforceps placethecutfin into alabelled vial(containing 1.5mlethanol) Note:Alwayshandlethetissueusingsterilized toolsto avoidanycontaminations

13 Closethevial captightlyandplaceitinasafe container

14 Repeatsteps2to 13forthenextsample

11

7. TRANSPORTATION OFVIALSTOLABORATORY

Once 50 samplesarecollectedfrom one samplingsite, theymustbesecurelypacked into aboxforshipmentto Laboratoryasfollows:

i). Shipping requires draining of ethanol from vials (please ensure that the tissue is still maintained in a wet form), or alternatively replace ethanol with non-combustibleDMSO solution—ifavailable

ii). Place the vials in a sample box and wrap the sample box with air bubble plastic provided and seal the airbubble plastic with cellophane tape and in a postagebox(provided)

Atechnical officeris requiredto sendall thesamplesto Laboratoryusingcourierservice (e.g. DHL,FEDEX,etc.).

REFERENCES

SEAFDEC/MFRDMD. 2015. Standard Operating Procedure for data collection and analysis oftheneritictunas.

“Tuna Identification Sheet” posterpublished from Project on Information Collection of HighlyMigratorySpecies inSoutheastAsian Waters: Tuna (2008-2012)

12

APPENDIXI:Form 1

GeneticStudyforLongtail Tuna(Thunnustonggot)Stock inthe SoutheastAsian Region Form 1:FishSamplesCollection

Country:

Sampling site:

Date:

Species:

Typeoffishinggear:

Vesselcategory/Fishingzone:

No. ofSamples:

Plastic Ref.No:

Cutthispartandputintheplasticbag

*Additionalformisneededifmoresamplesarerequired

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APPENDIXII:FlowChart

FlowChartforTissueSampleCollectionProcedure START

(Fish samplingatport/samplingsite)

FILL UP FORM 1, PUT THE CUT NUMBER INSIDE

PLASTIC BAG PLACE SAMPLES INTO

PLASTIC BAG

PUT PLASTIC BAG SAMPLES INTO ICE BOX

SEND TO LAB OR

(Tissuesamplecuttingandpreservation)

KEEP IN REFRIGERATOR -20°C

COPY INFO FROM FORM 1 TO FORM 2

LABEL AND FILL UP EACH VIAL WTH 95% ETHANOL WIPE FISH WITH TISSUE PAPER

WASH FORCEP &

SCISSORS WITH CLEAN WATER WEIGH, MEASURE, CAPTURE

PHOTO OF EACH FISH AND RECORD INTO FORM 2

CUT FISH SECOND DORSAL FrN

USING SCISSORS WASH FORCEP &

SCISSORS WITH 95%

ETHANOL PLACE IT IN A VIAL USING

FORCEP

SCREW THE VIAL CAP TIGHTLY

PACK&SENDTO SEAFDEC/MFRDMD

m

SEAFDEC/IV

SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER