PRELIMINARY MORPHOLOGICAL AND MOLECULAR WORK … · menurun kerana penangkapan ikan berlebihan dan...

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PRELIMINARY MORPHOLOGICAL AND MOLECULAR WORK ON SEAHORSES (genus Hippocampus)

FROM JOHOR

Nurul Jannah Binti Ismail

QL 638 Bachelor of Science with HonoursS9 (Aquatic Resource Science and Management)N974 20121012

Pusat KJtidmat MakJumat Akademik UNlVERSm MALAVSIA SARAWAK

Preliminary morphological and molecular work on seahorses (genus Hippocampus) from Johor

PKHIDMAT MAKLUMAT AKADIMIK

111111111 fOnlllllllll1 1000235657

Nurul Jannah binti Ismail (24748)

A report submitted in partial fulfilment of requirement for degree of Bachelor of Science with Honours

Supervisor Dr Ruhana Hassan

Aquatic Resource Science and Management Department of Aquatic Science

Faculty of Resource Science and Technology Universiti Malaysia Sarawak 2012

Pusat khidD1~t Maklumat Akademik imiddot

UNlVERSm MALAYSIA SARAWAK

Table of Contents

Acknowledgement

Declaration

List of abbreviation

List of Figures

List of tables

ABSTRACT

10 INTRODUCTION

20 LITERATURE REVIEWS

21 Classification of seahorse

22 The importance of seahorse

23 Feeding habit of seahorses

24 Genetics studies on seahorse

MATERIALS AND METHODS

31 Sample collection

32 Morphological identification

33 Laboratory work

331 Preparation ofbuffer solution

332 Total genomic DNA extraction

333 Gel Electrophoresis

334 Gel documentation of DNA bands

335 Polymerase chain reaction (PCR)

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334 PCR product purification 20

335 DNA sequencing 20

336 Data analysis 20

40 RESULTS AND DISCUSSION

41 Morphology measurement 21

42 Total genomic DNA extraction 33

43 Polymerase Chain Reaction (PCR) 36

44 Sequencing data analysis 39

45 Genetic divergence 42

46 Phylogenetic analysis 44

50 CONCLUSION AND RECOMMENDATION 47

60 REFERENCES 48

70 APPENDICES 51

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ACKNOWLEDGEMENT

AlhamdulHlah fullest gratitude to the Almighty for His blessings the faith and courage given

I was able to complete my final year project Firstly I would like to express my deep thankful

to my supervisor Dr Ruhana Hassan for all her wonderful insight and supervision throughout

the duration of thjs project A lot of thanks also goes to Madam Rosmaria Abu Darim and

entire staffs of Fisheries Research Institute (FRI) Gelang Patah lohor for their willingness to

help me in order to obtain sample of seahorses for this project I also grateful for guidance and

assistance of post graduate students especially Ms Nurhartini Kamalia Yahya Ms Farah

Adibah Esa Ms Nursyuhaida Md Shahid Mr Mohd Izwan Zulaini and Aquatic lab assistants

for their good cooperation and assistance in completing this project My appreciation also goes

to my best coursemate Tuan Zuraida Tuan Hussin for her willingness to accompany me in the

laboratory especially at night Besides from that I would like to thank to my fellow friends

especially Christine Anthonius Siti Rokhaiya Biollah Nur Hazwanie Izyan Mohd Nizam

Noor Liyana Osman and lunirah lamil and entire final year students of Department of Aquatic

Science for their continuous help and supports in making this project a success Last but not

the least my hearties gratitude goes to my parents Mr Ismail Hassim and Madam Soliah

Tamon and the rest of my family members for their amazing love and support in

accomplishing this project Thank you very much for being patient and supportive whatever I

do during my study

bull

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an

application for another degree qualification of any other university or institution of higher

learning

NURUL JANNAH BINTI ISMAIL

Aquatic Resource Science And Management

Department of Aquatic Resources

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol

Deoxynuc1eotide triphosphate

Cetyl Trimethyl Ammonium Bromide

Polymerase chain reaction

Mitochondrial deoxynuc1eic acid

Restriction fragments length polymorphisms

Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid

National Center for Biotechnology Infonnation

Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46

External morphology of sea horses (adopted from Lourie et aI 2004) 5

Mitochondrial DNA that involve in the molecular identification of 10 seahorse Map of the sampling site 12

Height (Ht) and standard length (SL) as measured on a seahorse 13

Simplified flow of work of total genomic DNA extraction for seahorse 16

peR profile (adopted from FitzSimmons et al 1997) 19

Sample of seahorse (KL 1) 22

Sample of seahorse (KL2) 24





Gel electrophoresis photograph showing total genomic DNA extraction 34 products Electrophoresis analysis of the cytochrome b peR product obtained from 37 KL2 Data chromatograms for peR product of seahorse (genus Hippocampus) 40

BLAST result of cyt b gene sequence obtained from KL2 and Hkuda 41 (AY422137) Neigbour Joining (NJ) phylogenetic tree 45

Maximum Parsimony (MP) tree 46

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List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42

Taxonomy of the seahorses Hippocampus following Kuiter (2000) 4

Important Morphological Terms (adopted from Lourie et ai 2004) 6

2X CT AB buffer recipe for 500ml stock 14

Measurement of selected morphological features for seahorse KL 1 23

Measurement of selected morphological features for seahorse KL2 24





Pairwise distance (Kimura 2- parameter) between Hippocampus spp 43

v


Nurul Jannah binti Ismail

Aquatic Science Resource and Management Programme Faculty of Resource Science and Technology


ABSTRACT

Seahorse (Hippocampus spp) is a protected animal as stated in Appendix II Convention ofInternational Trade of Endangered Species (CITES) However illegal trade of seahorse still exist due to high demand in traditional Chinese medicine (TCM) and ornamental businesses Population of seahorses is declining worldwide due to overfishing and other factors Conservation efforts have been difficult due to difficulties in identifying the species of seahorse This study is designed to document detail morphology of seahorses from lohor and to sequence the partial cytochrome b (cyt b) gene in order to detennine the genetic diversity and molecular phylogeny of the seahorses from lohor Seahorse samples were donated by Fisheries Research Institute (FRI) Gelang Patah lohor A total of 6 seahorses were subjected to identification based on morphological characteristics In addition molecular work involved total genomic DNA extraction PCR to amplify partial cyt b gene direct DNA sequencing and data analysis Based on morphological characteristics KL1 is H kuda KL4 is H spinosissimus KL5 is H subelongatus and KL6 is H histrix Identification of the other 2 seahorse samples was unresolved A total 735bp partial cyt b gene sequences was obtained from seahorse KL2 and it matched with sequence of H kuda accession number of AY422137 cyt b gene sequence This partial cyt b gene had been submitted to GenBank and its accession number is lX 217831 Based on partial cyt b gene sequence analysis KL2 and other H kuda fonned a clade with high bootstrap value therefore H kuda is monophyletic Within H kuda samples involved in this study genetic divergence value is between 01 - 56 indicating intraspecific variation between samples

Keyword seahorses (genus Hippocampus) morphological characters partial cytochrome b (cyt b) gene bootstrap value genetic divergence

ABSTRAK

Kuda laut merupakan salah satu daripada haiwan yang dilindungi seperti yang dinyatakan dalam Lampiran 2 Konvensyen Perdagangan Antarabangsa terhadap Spesis Terancam (CITES) Walau bagaimanapun perdagangan haram terhadap spesis ini masih wujud dan berleluasa kerana permintaan yang tinggi terutama dalam perubatan tradisional Cina dan perniagaan haiwan hiasan Di peringkat global populasi spesis ini terus menurun kerana penangkapan ikan berlebihan dan faktor- faktor lain Usaha- usaha pemuliharaan terhadap kuda laut sukar dijalankan kerana kesukaran dalam mengenalpasti spesis kuda laut Kajian ini dijalankan bertujuan untuk mendokumentasikan moifologi kuda laut dari Johor Selain itu objektif kajian inii juga adalah untuk mendapatkan sebahagian turutan gen cytochrome b (cyt b) yang digunapakai dalam menentukan kepelbagaian genetik kuda laut dari Johor Sampel kuda kaut untuk kajian ini telah disumbangkan oleh Institut Penyelidikan Peri kanan Gelang Patah Johor Kesemua 6 ekor sampel kuda laut telah dikenalpasti berdasarkan ciri- ciri moifologi yang terdapat pada sampel tersebut Kerja- kerja molecular melibatkan pengekstrakan DNA PeR pengaturan DNA secara langsung dan analisis data Berdasarkan karakter moifologi KLI adalah kuda KIA adalah H spinosissimus KL5 adalah H subelongatus dan KL6 adalah H histrix manakala 2 sampel kuda laut yang lain tidak dapat dikenalpasti secara tepat Sebahagian turutan gen cyt b sepanjang 735bp telah diperoleh dari kuda laut KL2 dan ia berpadanan dengan turutan H kuda (no rujukan AY422137) cyt b gen Turutan gen cyt b telah dihantar ke GenBank dengan no rujukan JX217831 Berdasarkan analisis daripada sebahagian turutan gen cyt b KL2 dan H kuda yang lain telah membentuk satu klad yang mempunyai nilai bootstrap yang tinggi jadi H kuda adalah monofiletik Sampel H kuda yang terlibat dalam kajian ini mempunyai nilai percapahan genetik antara OlYrr 56 yang menunjukkan variasi intraspesijik antara sampel

Kata kunci kuda laut (genus Hippocampus) karakter moifologi sebahagaian gen cyt b nilai bootstrap variasi genetik

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Chapter 1

10 Introduction

During the 1 i h meeting of the Conference of Parties to the Convention on International Trade

in Endangered Species of Wild Fauna and Flora (CITES) all species of seahorses (genus

Hippocampus) have been included into Appendix II (Lourie et al 2004) Flora and fauna

which in Appendix II are considered as species those are or might become threatened by

international trade The trade is managed for sustainability which means the exports require

non detriment findings

Seahorses are grouped with pipefishes pipehorses and seadragons in the family Syngnathidae

There are more than 200 species in this family which are 35 of them are seahorses (Lourie et

al 2004) There are some characters that be used to distinguish seahorses from other members

like a prehensile tail the absence of the caudal fin the position of the head at a right angle to

the trunk a brood pouch sealed along the midline and a raise dorsal fin base (Fritzsche 1980)

In addition seahorses have some remarkable adaptations to differentiate them with other

aquatic organisms such as hard bony armor on the body a prehensile tail that can be used for

gripping binocular vision excellent camouflage and an unusual form of reproduction

Seahorses are found throughout the tropical and temperate regions of both the Atlantic and

Indo- Pacific biomes but their origin and evolutionary history are not well understood

Besides only a few research regarding seahorses have been conducted For example Teske et

(2003) had conducted study on the evolutionary history of seahorses (Syngnathidae

Hippocampus) from West Pacific origin and Atlantic Ocean and molecular identification of

seahorses for sale in the shops in California by Sander et al (2007) Besides seahbrses are

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also threatened by direct exploitation accidental capture in non- selective fishing gear and

degradation of the habitats In Traditional Chinese Medicine (TCM) seahorses and its

derivatives become one of the largest consumption in the medicine

Although seahorses exist in Malaysia only some of Malaysian aware about their existence in

Malaysian coastal waters Several websites like Save Our Seahorses (wwwsosmalaysiaorg)

highlighted some conservation activities related to seahorses in Malaysia Up to date there is

no data on nucleotide of seahorses from Johor deposited in National Centre for Biotechnology

Information (wwwnchinlmnihgov) Several studies regarding molecular work on seahorses

had been conducted in other countries such as United States South Africa and Canada and had

been successful in aiding the identification of seahorses (Lourie et at 2004 Sanders et at

2007) However there is no detail of where the samples come from Therefore this study is

designed to sequence and analyse the cytochrome b gene of seahorses from Johor

The objectives of this study are

1 To document det~l morphology of seahorses from JohoL

11 To sequence the cytochrome b (cyt b) gene from Hippocampus spp from Johor

iii To determine the genetic diversity and molecular phylogeny of the seahorses

~

3

- - - ---

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Chapter 2

20 Literature Review

21 Classification of seahorses

Seahorses belong to the genus Hippocampus of the family Syngnathidae This family consists

of 55 genera of pipefishes pipehorses and seadragons (Kuiter 2000) The family

Syngnathidae falls within the order Gasterosteiformes Detail taxonomy hierarchy of the

seahorse is shown in Table 21 below

Table 21 Taxonomy of the seahorses Hippocampus following Kuiter (2000)

- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~

Subphylum Vertebrata

bull - r _ - - bullbull ~-_ ~_ ~ ~~~ - -- bull _ bull I r

- -- -~

Class Osteichthyes (bony fishes)

Infraclass Neopterygii --- - - -- -_ - --3~- ~-~ I bull ~

Order

Genus

- - - - - --- -~ ------Gasterosteiformes

-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

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oPusat Khtdmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

In order to identify seahorse there are some important parts (morphological characters) such

coronet cheek spines pectoral fin and dorsal fin Detail external characters of the seahorse as

in Figure 21 and Table 22

l~

coronet

pectoral fin lyesPine nose spine

A ~nout 1

trunk

dorsal fin

anal fin

first ring tail last trunk ring

tail brood pouch (males)

Fig 21 External morphology of seahorses (adopted from Lourie et ai 2004)

5

Table 22 Important Morphological Tenns (adopted from Lourie et al 2004)

Cheek spines (CS) Spines at the bottom of the operculum on each side of the animals head

Coronet Enlarged structure found on the top of the head of some species

bull -- - - - ~~---- ~~ ~ J bull ~bullbullT

- shy- - --- - - ~~~ shy

Eye spines (ES) Spines directly above the eye

-rmiddot-middot~p-~-rrmiddot-middot~ lt ~ ~ ~~~~-

shy

Height (Ht) Distance between of the tip of the coronet to the tip of the uncurled tail

Nose spine Single spine located in front of the eyes on the upper side of the snout in some species

Pectoral rm rays

Tail length

Trunk length

Tubercles

Bones that support the pectoral fin

Distance between the lateral mid- point of the last trunk ring to the tip of the uncurled tail

Distance from the mid- point of the cleithral ring to the lateral mid- point of the last trunk ring

Raised rounded nodules located at the intersections of rings and ridges (some species only)

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22 The importance of seahorses

221 Ecology

Seahorses playa vital role in marine food web as secondary consumer They can co- exist with

many species of shrimps and other bottom feeders Seahorses have ability to camouflage They

are capable of changing their body colors ranging from ash grey orange brown yellow red

and black to blend into their environment (Lourie et at 2004) This ability give advantage to

seahorse to hide easily from predators and increase success of predation Seagrass becomes

favourable habitat to the seahorses as they use the blade of seagrass as holdfast

222 Socio- economy

Every year it is estimated about 20 million individual seahorses enter the internartional

market Most of them are dried and sold for traditional Chinese medicine (TCM) According

to Vincent (1996) some of seahorses are also being sold or live in aquarium trade as curios

Seahorses may generate some income to the poorest fishers in developing countries They

become critical targets as source of daily income due to high demand and their high market

value In some Sotuh ~ast Asia countries seahorses are believe to give powerful tonic

especially to the men They are also use as herbs in Chinese soup which able to refresh the

body and mind as well as to handle stress

7

23 Feeding habit of the seahorses

Seahorses are ambush predators and consume primarily live and mobile prey types (Warland

2003) They win wait their prey comes nearer to them and then they used their long snout to

drawn up them with rapid intake of water The eyes of seahorses move independently which

enable them to maximize its search area or monitoring region According to Woods (1998)

small organisms like small crustaceans such as amphipods fish fry and other invertebrates will

be ingested by seahorse using snouts Seahorses may change their diet ontogenetically Woods

(2002) who studied on the gut content of the seahorses indicates that the smaller individuals of

seahorses (952- 1375cm Ht) feed on the amphipods whereas the larger seahorses (gt 1384cm

Ht) consumed more on shrimp

8

species analysis

24 Genetic studies on seahorses

Most of population genetics studies on seahorses Hippocampus spp were carried out from the

West Pacific and the Atlantic Ocean (Teske et al 2003) The analysis of mtDNA is a robust

tool for characterization and verification of seahorses especially in the trade that involved

them Casey et al (2004) stated that cyt b divergence among seahorse species is relatively high

However in order to distinguish between genetically similar species of seahorses like Hreidi

and Halgiricus additional sequence data are required The capability of population- level

identification can be affordable via molecular analysis which is more useful than simple

In some large island nation such Indonesia and Philippines they usually harbor genetically

distinct regional populations of certain seahorse species (Lourie et aI 2005) The study of

intraspecific variations in cyt b and control region sequences for Hingens by Sander et al

(2007) had revealed that there is low genetic variation among H ingens which may make

phytogeographic identification of the origin difficult

Based on the data available in GenBank there are 1438 nucleotide sequence belonged to

seahorses namely (accession numbers DQ912699 - DQ912786) There are sequences for

species with available phylogeographic data such as H barbouri (A Y 495716- A Y 495738) H

spinosissimus (A Y 495739- A Y 495825) and H trimaculatus (AF192699- AF192703)

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Seahorses have a low rate of control region evolution compared to other aquatic organisms

For some species like H ingens the ratio of transit it on transversion changes in control region

sequences is high (60) shows that low evolutionary rate might not limit seahorse control

region variability (Sander et ai 2007) Comperative phylogeography has the potential to give

patterns and processes in order to determine species distribution and help inform conservation

decisions In Southest Asia all species of seahorses are restricted in their dispersal capibilities

to some region

In the phylogenetic studies cyt b is used to determine relationships between organisms due to

its sequence variablity It is useful in determining relationship within families and genera for

species identification Cyt b gene is situated between D- loop and NDs in mitochondrial DNA

(mtDNA) as in Fig 22

Fig 22 Mitochondrial DNA that involve in the molecular identification of seahorse (adopted from httpwwwnaturecomcrjoumalv 1 7 n3images)

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Chapter 3

30 Materials and Methods


The seahorse specimens used in this study were collected by other group of researchers in

Fisheries Research Institute (FRI) Gelang Patah Johor in July 2011 at Pulai River Estuary

Johor Bahru Johor (Figure 31) This study involved seahorse specimens which donated by

FRI Researchers Whole carcasses of seahorses have been immersed in the 70 ethanol and

ethylenediaminetetraacetic acid (EDT A) and the samples were sent to Aquatic Molecular

Laboratory via postage Specimens were stored on working bench at room temperature

11

Pulai River Estuary

Fig 31 Map of the sampling site in this study

12


Samples of seahorse obtained were measured up to the nearest mm using thread ruler

magnifying glass and calculator The height (Ht) the head length (HL) and snout length (SnL)

were recorded up to nearest em The HLlSnL ratio of the specimen were calculated and

recorded The number of tail rings (TaR) number of trunk rings (TrR) of the dorsal fin and

pectoral fin rays were counted and recorded Identification of seahorse up to species level was

referred to Lourie et al (2004) in Figure 32

Head length

Height

Standard length Tail (=head length +length curved trunk length + tail length)

Fig 32 Height (Ht) and standard length (SL) as measured on a seahorse (Lourie et ai 1999)

13

33 Laboratory work

331 Preparation of buffer solution for modified eTAB (Doyle and Doyle 1987)

(a) CTAB (cetyl- trimetyhl ammonium bromide)

Table 31 2X CT AB buffer recipe for 500mI stock

Chemicals Molecular weight (g) Quantity needed (g)

- -c- -----r~

~l Cetyl- trimethylammonium 36409 1000

bromide (CT AB)

Using analytical balance all chemical except 2- mercaptoethanol ~- mercepto were weighted

and put into 500mI beaker Then 400ml of ddH20 was added to dilute all salts under hot plate

The solution was stirred using magnetic stirrer until the crystal clear solution is formed The

solution was left to cool down and then IOOIlI of 2- mercaptoethanol ~- mercepto was added

to the solution in the fumeboard Next the solution was transferred into 500mI volumetric

JIask filter and ddH20 was added until the total volume reach 50Oml The solution then was

middotWftlftNL1 with aluminium foil to prevent light penetration because CTAB is light- sensitive

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Total genomic DNA was extracted using modified Cetyl- trim ethyl Ammonium Bromide

(CTAB) method (Doyle and Doyle 1987) with the presence of Proteinase K A total of 700111

of 2X CT AB Buffer and 5 III Proteinase K was added to appendorf tube containing minced

parts (head tailor pouch) of Hippocampus spp Then the tube was incubated in Protech

model 903 incubator at 65deg C for up to five hours Then 700111 of Chloroform Isoamyl

Alcohol (241) was added and was centrifuged using refrigerated high speed micro centrifuge

CF 15RX Hitachi RX series at 15000 rotations per minutes (rpm) for 10 minutes at 4degC The

supernatant was transferred and 500111 of absolute ethanol (EtOH) was added The mixture

then was incubated in -20deg C for 15- 30 minutes or overnight The mixture was centrifuged

again at 15000 rpm for another 10 minutes at 4degC A total of 500111 of 70 EtOH was added to

the mixture After that the tubes were centrifuged at 15000 rpm for 10 minutes at 4degC The

supernatant was discarded and pellet was air- dried in room temperature Pellet then was

dissolved in 50111 of sterile distilled water (dH20) and stored in the freezer at -20deg C for future

usage Summary of the work is simplified in Figure 33

15

Pusat KJtidmat MakJumat Akademik UNlVERSm MALAVSIA SARAWAK


PKHIDMAT MAKLUMAT AKADIMIK

111111111 fOnlllllllll1 1000235657

Nurul Jannah binti Ismail (24748)

A report submitted in partial fulfilment of requirement for degree of Bachelor of Science with Honours

Supervisor Dr Ruhana Hassan

Aquatic Resource Science and Management Department of Aquatic Science

Faculty of Resource Science and Technology Universiti Malaysia Sarawak 2012



Table of Contents

Acknowledgement

Declaration


List of Figures

List of tables

ABSTRACT

10 INTRODUCTION









33 Laboratory work






Page

11

111

IV

V

1

2

4

7

8

9

11

13

14

15

17

18

19

p












60 REFERENCES 48

70 APPENDICES 51

i middot

ACKNOWLEDGEMENT


















do during my study

bull

DECLARATION



learning






ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















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List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

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Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

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to some region






10

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Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

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15



Table of Contents

Acknowledgement

Declaration


List of Figures

List of tables

ABSTRACT

10 INTRODUCTION









33 Laboratory work






Page

11

111

IV

V

1

2

4

7

8

9

11

13

14

15

17

18

19

p












60 REFERENCES 48

70 APPENDICES 51

i middot

ACKNOWLEDGEMENT


















do during my study

bull

DECLARATION



learning






ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

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List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

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to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

p












60 REFERENCES 48

70 APPENDICES 51

i middot

ACKNOWLEDGEMENT


















do during my study

bull

DECLARATION



learning






ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

i middot

ACKNOWLEDGEMENT


















do during my study

bull

DECLARATION



learning






ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

DECLARATION



learning






ii


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15


~l

rpm

mm

ml

mM

EtOH

dNTPs

CTAB

PCR

mtDNA

RFLPs

NaCI

MgClz

bp

DNA

NCBI

UV

ng

Microliter

Round per minutes

Millimetre

Millilitre

Millimole

Ethanol






Sodium chloride

Magnesium chloride

Base pair

Deoxynuc1eic acid


Ultra violet

nanogram

iii

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

~

List of figures

Caption Page number

Fig 21

Fig 22

Fig 31

Fig 32

Fig 33

Fig 34

Fig 41 (a)

Fig41(b)

Fig 41 (c)

Fig 41 (d)

Fig 41 (e)

Fig4l(f)

Fig 42

Fig 43

Fig 44(a)

Fig44(b)

Fig 45

Fig 46















iv

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

imiddot

List of tables

Caption Page number

Table 21

Table 22

Table 31

Table 41 (a)

Table 41 (b)

Table 4I(c)

Table 41 (d)

Table 41 (e)

Table 41 (t)

Table42











v





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15





ABSTRACT



ABSTRAK



1

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

al

imiddot

Chapter 1

10 Introduction





















2

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

imiddot

















~

3

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

- - - ---

i middot

Chapter 2








- 0 _ ___ ___ -_ ~ ~ ~~ ~ __ ~ ___- ~-- -_~~~~~



- -- -~



Order

Genus


-- - - - ~ ~---~~~

Hippocampus

- - - - - - _- - -------- - -- ~-- I - _ ---bullbull-- --~- ----- ~

I 4

4





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15





l~

coronet


A ~nout 1

trunk

dorsal fin

anal fin




5





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15





- shy- - --- - - ~~~ shy



shy



Pectoral rm rays

Tail length

Trunk length

Tubercles





6

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

imiddot


221 Ecology







222 Socio- economy









7











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15











8

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

species analysis


















9

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

i middot







to some region






10

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

imiddot

Chapter 3









11

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

Pulai River Estuary


12








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15








Head length

Height



13

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

33 Laboratory work





- -c- -----r~


bromide (CT AB)








14

imiddot
















15

imiddot
















15

PRELIMINARY MORPHOLOGICAL AND MOLECULAR WORK … · menurun kerana penangkapan ikan berlebihan dan...

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Transcript of PRELIMINARY MORPHOLOGICAL AND MOLECULAR WORK … · menurun kerana penangkapan ikan berlebihan dan...