· ., ".

YELLOW PUFFERFSIH OF1 SATANG SA,RIBAS, Si\.RA\VA.K

Azimah Binti Apendi (29701)

QL Bachelor of Science with Honours638

(Alluatic Resource Science and Management)T32 2014A995

2014

Yellow Pufferfish of Batang Saribas, Sarawak

Azimah binti Apendi (29701)

Supervisor: Dr. Ruhana Hassan

Aquatic Science Resource and Management Programme

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak 20 June 2014

DECLARATION

I hereby declare that the work in this project is my own except for quotations and

summaries which have been duly acknowledged. No portion of the work referred to

this dissertation has been submitted in support of an application for another degree

qualification of this or any other university or institution of higher learning.

......................................

Azimah binti Apendi (29701)

Aquatic Resources Science and Management Programme

Faculty of Resources Science and Technology

Universiti Malaysia Sarawak

i

Acknowledgement

Alhamdulillah, my greatest gratitude to Allah with His power, I was able to

complete my Final Year Project “Yellow Pufferfish of Batang Saribas, Sarawak”.

First and foremost, my deepest gratitude to my supervisor Dr.Ruhana Hassan for

her continuous support and guidance. For without her, I would have been lost during this

study.

I would also like to thank postgraduate students, Kak Tini, Abg Khai, Kak Sue,

Kak Wani and Abg Izwan for guiding and helping me from with lab works and their

endless encouragement. Not to forget, my labmates Natasha Arina, Natasya, Noor Hidayu,

Amirul and fellow coursemates accompanying me throughout this study. and all of my

course mates for their continuous encouragement and help throughout this study.

My greatest gratefulness to my mother, Saloma binti Gapor who had always been

there for me and gives me strength whenever I need them the most. Not to forget my

wonderful siblings Zulfadli, Idhwan, Nur Azimah and Ijan, thank you so much. Thanks to

Ahmad Akhmal for his continuous supports through my tears. I would also like to thank

my motivation team, Ct, Kin, Elly, Fain, Ena, Mel, Nora, Faiha and Fazzrul for everything.

My deepest gratitude to all lecturers and staffs from Department of Aquatic for

their assistance. Also to my loving grandfather, Hj Gapor for his wisdom and for being my

personal tour guide during sampling. The people of district Betong who had participated in

this study, thank you. Lastly, I would like to thank Faculty of Resource Science and

Technology for the opportunity to carry out this project. Thank you all.

ii

Table of Content

Acknowledgement……………………………………………………………..

i

Table of contents………………………………………………………………… ii

List of abbreviation……………………………………………………………….

iv

List of figures……………………………………………………………………..

v

List of tables……………………………………………………………………...

vi

Abstract………………………………………………………………….............

1

1.0 Introduction…………………………………………………………………..

2

2.0 Literature Review…………………………………….…………….………….

2.1 Taxonomy………..…………………………….…………...………....

2.2 Yellow Pufferfish…………..…………………...…….…….…………

2.3 Morphological characters……….……………...……….….………….

2.4 Difference between genus Xenopterus and Chonerhinos.….…..……..

2.5 Cytochrome b……………………….…….……………………………

2.6 Studies on Yellow Pufferfish………………..……….....…………….

2.7 Molecular Biology Techniques……………………………………….

2.7.1 Total Genomic DNA Extraction…………………………….

2.7.2 Polymerase Chain Reaction (PCR)………………………....

2.8 Socio-economic of Locals…………………………………………….

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5

5

6

7

8

9

10

10

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3.0 Materials and Method………………………………………………………....

3.1 Study site……………………………………………………………....

3.2 sample Collection…………………….…………………………….….

3.3 Morphological assessment…………………………………………….

3.4 Total Genomic DNA Extraction……………...……………………….

3.5 Optical Density Reading………………………………...……………..

3.6 Polymerase Chain Reaction …………….……………………………..

3.6.1 Cytochrome b gene………………………………………….. 3.6.2 Cytochrome Oxidase I gene…………………………………….

3.6.3 16S RNA gene……………………………………………………

3.8 Agarose Gel Electrophoresis…................………………...…………... 3.9 Socio-economic Study……………………………………………………….

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4.0 Results and discussions………………………………...…………………….

4.1 Morphological Assesment………………………..…………………..

4.2 Preliminary Genetic Analysis……..……………...………………..… 4.2.1 DNA Extraction……………………………………….……………

4.2.2 Polymerase Chain Reaction..……………………………….

4.3 PCR Optimization……………………………………...………………

4.3.1 Cytochrome b gene…..…………………………………...

4.3.2 COI gene…………………………………………………..

4.3.3 16S RNA gene……………………………………………

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21

24

25

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32

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iii

4.4 Socio-economic Profile………………………………………………

35

5.0 Conclusion and Recommendations.....................................................................

5.1 Conclusion……………………………………………………………..

5.2 Recommendations……………………………………………………..

38

38

38

6.0 References…………………………………………………...…………………

39

7.0 Appendix……………………………………………………………………….

44

iv

List of Abbreviations

DNA Deoxyribonucleic acid

mtDNA Mitochondrial DNA

PCR Polymerase Chain Reaction

Cyt b Cytochrome b

COI Cytochrome Oxidase I

CTAB Cetyltrimethylammonium Bromide

EtOH Ethanol

MgCl2 Magnesium chloride

dNTP Deoxyribonucleic Triphosphate

ddH2O Deionized distilled water

EtBr Ethidium bromide

v

List of Figures

Title

Pages

Figure 2.1 Taxonomy of Pufferfish (Source: Nelson, 2006)

5

Figure 2.2 mtDNA gene map (www.ufrgs.br, retrieved on 02/10/13)

9

Figure 3.1 Sampling site, Batang Saribas, Sarawak (N 1°24’0”, E

111°31’0”)

13

Figure 3.2 Illustration of pufferfish showing total length(TL), standard

length(SL), dorsal fin and anal fin (Santini et al., 2013a)

14

Figure 3.3 PCR thermal profiles using cyt b gene (Sevilla et al., 2007) 17

Figure 3.4 PCR thermal profiles using COI gene

18

Figure 3.5 PCR thermal profiles using 16S RNA gene

19

Figure 4.1 Xenopterus naritus, A,31-32; D,26-27

22

Figure 4.1.a Teeth of yellow pufferfish

22

Figure 4.1.b Mouth of yellow pufferfish

23

Figure 4.2 Agarose gel electrophoresis photograph showing the first DNA

extraction products of XnBS01, XnBS02, XnBS03 and

XnBS03 using modified CTAB protocol by Doyle and Doyle

(1987).

26

Figure 4.3 Agarose gel electrophoresis photograph showing the second

DNA extraction products of XnBS01with XnBS02, XnBS03

and XnBS03 using modified CTAB protocol by Doyle and

Doyle (1987)

26

Figure 4.4 Yellow pufferfish sold in Betong were either fresh, salted/dried

or fermented form

36

vi

List of Table

Title

Page

Table 4.0 Voucher numbers of yellow pufferfish individuals and

preservations

21

Table 4.1 Meristic data of X. naritus collected from Batang Saribas

23

Table 4.2 Morphometric data of X. naritus collected from Batang Saribas

24

Table 4.3 Optical density reading for DNA extraction of yellow

pufferfish

27

Table 4.4 Primers used in this study

28

1

Yellow Pufferfish of Batang Saribas, Sarawak

Azimah binti Apendi

Aquatic Resource Science and Management Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Abstract

Yellow pufferfish or Xenopterus naritus (Tetraodontiformes: Tetraodontidae) is a brackish water or estuarine

species. In Malaysia, the species can be found abundantly in the state of Sarawak and a well-known species

in Batang Saribas, Sarawak from June to September. The species has economic value to the local people and

ecological value on the mangrove and estuarine area of Batang Saribas. There are several studies that had

been done on yellow pufferfish, but the molecular aspects of this species in Batang Saribas is still lacking. A

total of 4 male yellow pufferfish were collected and examined. All the yellow pufferfish were yellowish in

color and torpedo-like shape. The standard length was 14.85±0.191 cm and the total length was 18.33±0.583 cm. The number of soft dorsal fins was 31-33 and the soft anal fins were 26-27. Total DNA extraction using

modified Doyle & Doyle (1987) was successful where only one individual showing pure DNA (OD260/OD230

~1.80). The PCR of cytochrome b, Cytochrome Oxidase I and 16S RNA genes were still at optimization

stage.

Keywords: Xenopterus naritus, Tetraodontiformes, cytochrome b, Cytochrome Oxidase I, 16S RNA

Abstrak

Buntal kuning atau Xenopterus naritus (Tetraodontiformes: Tetraodontidae) adalah spesies air payau. Di

Malaysia, spesies ini boleh dijumpai dengan banyak di negeri Sarawak terutamanya Batang Saribas, Sarawak dari bulan Jun hingga September. Spesis ini mempunyai nilai ekonomi kepada penduduk tempatan dan nilai

ekologi terhadap kawasan bakau dan muara Batang Saribas. Beberapa kajian telah dijalankan keatas buntal

kuning namun kajian molekular mengenai buntal kuning dari Batang Saribas masih kurang. Empat ekor

buntal kuning jantan telah diambil dan dikaji. Semua buntal kuning mempunyai warna kuning dibadan dan

berbentuk torpedo. Panjang standard adalah 14.85±0.191 cm dan jumlah panjang adalah 18.33±0.583 cm.

Bilangan sirip dorsal lembut adalah 31-33 dan sirip anal lembut adalah 26-27. DNA telah berjaya diekstrak

menggunakan Doyle & Doyle (1987) dan hanya satu DNA sampel yang tulen (OD260/OD230 ~1.80). PCR

menggunakan cytochrome b, Cytochrome Oxidase I dan 16S RNA gen masih pada tahap pengoptimuman.

Kata kunci: Xenopterus naritus, Tetraodontiformes, cytochrome b, Cytochrome Oxidase I, 16S RN

2

1.0 Introduction

Pufferfish is known as blowfish, swellfish, globefish and balloon fish (Torda et al.,

1973). The name arises from their body inflation abilities. Pufferfish inflate themselves

with air or water in presence of predator, making themselves difficult to be swallowed

(Mehmet, 2011). Pufferfish is classified into three families which are Tetraodontidae,

Triodontidae and Diodontidae. Family Tetraodontidae includes freshwater, brackish and

marine waters species. According to Oliveira et al. (2006), the family Tetraodontidae

consists of 185 species and 28 genera of pufferfish. Most pufferfish are found abundantly

in warm, shallow tropical and sub-tropical water of west India, Japan, China, Phillipines,

Mexico, Taiwan and Southern Asia.

Pufferfish diet varies between species. Some species are carnivorous, some

herbivorous or omnivorous. They have outer jaws that are modified into strong, beak-like

structures which allow them to feed on corals and invertebrates (Moyle and Cech Jr.,

2004). Apart from their ability to inflate themselves, pufferfish are also famous for their

toxicity. Fozzard and Lipkind (2010) stated in their paper that tetrodoxin (TTX) is a small

molecular weight guanidinium neurotoxin that occludes voltage-gated sodium channels in

nerve and muscle tissue in this manner impending ion conductance. The name was taken

from pufferfish of family Tetrodontidae in which it was first found.

The yellow pufferfish, Xenopterus naritus is different from other puffers due to its

yellowish, bronze or golden colour especially towards the lower part of the body

(Gambang and Lim, 2004). Apart from their coloration, yellow pufferfish have a higher

3

numbers of anal, dorsal and pectoral fin rays compared to other puffers (Roberts, 1982).

The species can be found abundantly in the state of Sarawak in Malaysia especially in

Batang Sadong, Batang Lupar, Batang Krian and Batang Saribas. Atack (2006) reported

that Xenopterus naritus is a coastal and estuarine puffer species and can be found in many

brackish rivers in Sarawak.

Assessment on morphological characteristics of fish is done to identify the genus

and species (if possible). Although it has limitations compared to molecular data,

morphological characters are important for in-situ identification. Some of the examples of

morphological characters of family Tetraodontidae are, inflatable body, naked body or

short prickles usually confined to belly and absent of ribs and epineurals (Nelson, 2006).

However, when morphology is compromised, genetic information for example

mitochondrial DNA (mtDNA) gene sequences could be used to identify a species.

Compared to nuclear DNA, mtDNA has a high copy number (Carracedo et al., 2000) and

this allows it to be retrieved easily from trace elements.

There are few studies done on the molecular characteristics of pufferfish commonly

on genus Takifugu (Edwards et al., 1998; Venkatesh et al., 2000; Song et al., 2001; Kai et

al., 2005; Kai et al., 2011), genus Lagocephalus (Mohamad & Isa, 2013; Monaliza &

Mohamad, 2011), genus Tetraodon (Yamanoue et al., 2006; Yue et al., 2006), and others

(Brainerd, 1994; Tan, 2004; Nohan, 2004; Krumme, 2007; Nor Azman et al., 2013). These

studies include on morphology, genetic variation, population genetics of puffers and

toxicity.

4

A lot of studies on pufferfish had been done in the Indo-Pacific region such as

Japan and China on genus Takifugu. However, only few were done on yellow pufferfish or

Xenopterus naritus from Sarawak (Tan, 2004; Nohan, 2004; Nor Azman et al., 2013).

These studies are about the feeding habits, ecology, fecundity and toxicity of yellow

pufferfish in Sarawak. Thus, the information regarding morphology and molecular of

yellow pufferfish from Sarawak is still lacking. Therefore, this study is designed to

document morphology and molecular data for yellow pufferfish of Batang Saribas,

Sarawak. Besides that, yellow pufferfish are a known delicacy in Sarawak and there is an

annual yellow pufferfish festival in Batang Saribas near Betong. However, the socio-

economy of the locals associated with the processing or catchment of the yellow pufferfish

is still patchy. A pilot study is designed to properly document socio-economic profile of

local people involved in yellow pufferfish fisheries. The findings from this study could

become a baseline data for future studies related to yellow pufferfish in Sarawak.

The objectives of this study are to 1) document the morphological characters of

yellow pufferfish from Batang Saribas; 2) obtain and analyse Cyt b, COI and 16S RNA

genes from yellow pufferfish of Batang Saribas, Sarawak and 3) carry out a pilot study on

the socio-economy profile of the people in Batang Saribas involved in yellow pufferfish

fisheries.

5

2.0 Literature Review

2.1 Taxonomy

The taxonomy of yellow pufferfish is as shown as in Figure 2.1;

Kingdom: Animalia

Phylum: Chordata

Class: Actinopterygii

Order: Tetraodontiformes

Family: Tetraodontidae

Genus: Xenopterus

Species: X. naritus (Richardson, 1848)

Figure 2.1 Taxonomy of yellow pufferfish (Nelson, 2006)

2.2 Yellow pufferfish

Yellow pufferfish or commonly known as ‘Buntal Kuning’ locally are found

abundantly in the state of Sarawak in Malaysia. The fish is found inhabiting the brackish or

estuarine water (Roberts, 1982). According to Nor Azman et al. (2013), yellow pufferfish

is found in coastal waters near mangroves area particularly along Batang Saribas, Sarawak.

The species can be found from the estuary until the middle of the river where salinity is

6

above zero. In Sarawak, yellow pufferfish is a renowned species among the local people

who make yellow pufferfish as part of their delicacy. Yellow pufferfish is an anadromous

species where they migrate upstream, from a high salinity area to a lower salinity condition

prior ovulation to spawn (Nor Azman et al., 2013). According to Krumme et al. (2007),

tetraodontids are amongst the common fish sampled in tropical and subtropical mangroves.

Pufferfish may have significant ecological role in the mangrove areas, for their strong teeth

and jaw structure allow them to prey on fiddler crabs.

Pufferfish are famous for its toxicity and body inflation. According to Mehmet

(2011), pufferfish are probably the most common poisoning fish along the coasts of Asia

and there are about 120 species that live in the tropical seas which all belong to the order

Tetraodontiformes. Ellenhorn and Barceloux (1988) stated that TTX can be found in the

liver, gonads, intestines and skin of some the fishes of this order and are highly fatal. The

stomach of some Tetraodontiformes is highly modified to allow inflation (Nelson, 2006).

When in presence of prey or danger, they inflate themselves as protection (Brainerd, 1994)

by gulping water into a ventral diverticulum of their stomach. The absent of ribs and highly

extensible skin also allows the fish to change in size (Brainerd, 1994).

2.3 Morphological characters

Morphological characters aid in identifying the species of mammals, fishes and

even crustaceans. Yellow pufferfish name arose from the yellow or bronze coloration on its

body especially on the lower part of their body. Pufferfish can be distinguished from other

puffers by their number of fin rays. The species Xenopterus naritus has 32-38 dorsal fin

7

rays, 28-29 anal fin rays and 18-19 pectoral fin rays (Roberts, 1982). This species is said to

have an increased in number of fin rays compared to other pufferfish. Besides that, the

difference in number of teeth is distinctive between puffers which areone of the characters

used to determine their family. Tetraodontidae are a four-toothed puffers whereas

Diodontidae and Triodontidae are a two-toothed puffers and three-toothed puffers

respectively (Moyle and Cech Jr., 2004). The outer bones of their jaws are modified into

strong beak-like structures, which give them the appearance of teeth. Moyle and Cech Jr.

(2004) also reported that the body shape of the puffers in these three families are opposite

of sleek streamlining which are globular to pyramidal in shape with tiny gill openings,

small and paddle-like fins and a stiff, rounded tail.

2.4 Differences between genus Xenopterus and Chonerhinos

The genus Xenopterus and its close relative Chonerhinos had always been mistaken

as the same due to their close characters which are three lateral line canals on side of body,

dorsal fin with 22 or more rays, anal fin with 18 or more rays and absent of prefrontal

bones (Roberts,1982). Due to this, the yellow pufferfish was formerly known as

Chonerhinos naritus which was changed later to Xenopterus naritus. The genus

Xenopterus can be differentiated from genus Chonerhinos by their scales. In Xenopterus,

the scales are relatively large and extend dorsally towards the pectoral fin whereas in

Chonerhinos, the scales are relatively small and restricted to the head and body ventral to

the level of pectoral fin (Roberts, 1982).

8

These two genuses are highly specialized in the family Tetraodontidae due to their;

i) greater number of dorsal and anal fin rays compared to other tetraodontids, ii) an

elaborated lateral line system and iii) increased in scale sizes (Roberts, 1982). However,

Xenopterus are said to be more specialized because of its greater numbers of vertebrae and

fin rays along with its skull structures. The frontals of Xenopterus are more laterally

expanded and thickened which forms a large plate over most of the dorsal surface which

are difference from Chonerhinos skull which lacks prefrontal bones (Roberts, 1982).

Besides that, Xenopterus can only be found inhabiting the marine or estuarine waters while

Chonerhinos are found inhabiting the freshwater. The difficulties in distinguishing these

two genuses caused misinformation regarding the habitats of Xenopterus naritus which has

been said as a freshwater species.

2.5 Cytochrome b gene

Pufferfish is suitable to model the vertebrate’s genome due to its lack of repetitive

sequences (Mandrioli et al., 2000). The smallest vertebrate genomes yet measured belongs

to the smooth pufferfish of the family Tetraodontidae (Neafsey and Palumbi, 2003).

Therefore, comparing the genomes of different species of pufferfish with other organisms

will guide future approaches to the understanding of gene function, regulation and

evolution (Yamanoue et al., 2006). Genomes of pufferfish are uniquely compact (Brenner

et al., 1993) and contains unusually small introns and lack repetitive sequences and

pseudogenes (Song et al., 2001). A total of 20567 data on Tetraodontiformes can be found

on the Gene bank. There are studies on pufferfish of the Tetraodontiformes order using

Cyt b gene (Yue et al., 2006; Santinni et al., 2013a; Santinni et al., 2013b).

9

The most common genome used in species identification is cytochrome b (cyt b)

gene (Branicki et al., 2003) which had been used in phylogeny and forensic identification

(Pancorbo et al., 2004). According to Johns and Avise (1998), cyt b gene is chosen best

because of several reasons: 1) the gene is most extensively sequenced to date, 2) the

evolution of cyt b gene and the biochemistry of the protein products are better

characterized than other molecular system, 3) cyt b gene is phylogenetically informative.

The usefulness of cyt b gene analysis has been confirmed in identifying the biological

origin of samples (Pancorbo et al., 2004). The location of cyt b gene on mtDNA is shown

on the mtDNA gene map (Figure 2.2).

Figure 2.2 mtDNA gene map (www.ufrgs.br, retrieved on 02/10/13)

2.6 Studies on Yellow Pufferfish

There are limitations on information regarding yellow pufferfish from Sarawak.

Preliminary studies had been done by few graduates from Universiti Malaysia Sarawak

(Tan, 2004; Nohan, 2004) on feeding habits, ecology and fecundity of yellow pufferfish,

10

(Chen, 2008) on morphology and toxicity of yellow pufferfish and a preliminary study on

the molecular of yellow pufferfish (Ching, 2008). All three studies are on yellow pufferfish

from Batang Saribas. There is also one study on tetrodotoxin in yellow pufferfish from

Batang Saribas (Nor Azman et al., 2013) focusing on tetrodotoxin content in various

tissues of the yellow pufferfish.

2.7 Molecular Biology Techniques

2.7.1 Total Genomic DNA Extraction

Total genomic DNA extraction functions to isolate DNA or protein of

suitable integrity purity and quantity to allow subsequent analysis (Saunders and

Parker, 1999). Total genomic DNA can be extracted from different body parts with

different methods. In order to select the most appropriate DNA or protein

extraction, consideration should be given to the sample matrix, the target analyte

and type of analysis required prior to sample preparation (Terry et al., 2002).

Mandrioli et al. (2000) stated that DNA can be extracted from the liver cells of

Tetraodon fluviatilis, muscular tissues of pufferfish (Ishizaki et al., 2005) both

using phenol-chloroform techniques. DNA extraction from muscle tissue of the

genus Takifugu can be done by using phenol-chloroform and chloroform-isoamyl

method (Song et al., 2001).

11

2.7.2 Polymerase Chain Reaction (PCR)

Polymerase chain reaction is useful for amplifying small quantity of DNA

(Jones et al., 2007). This method allows amplification of DNA of endangered or

extinct species. According to Hartl and Jones (2000), PCR amplification requires

DNA polymerase and a pair of synthetic oligonucleotide primers that are

complimentary in sequence to the ends of the DNA sequence to be amplified. Few

factors that could optimize PCR amplification reaction include annealing

temperature, primer concentration, template concentration, MgCl2 concentration,

extension time and number of cycles (Grunenwald, 2003). In PCR reaction, the

primers should complement the DNA template perfectly (Dieffenbach and

Dveksler, 1995). If the forward and reverse primers do not complement the

template perfectly, the amplification will give an inaccurate result.In this study, the

PCR used is suitable to amplify cyt b, COI and 16S RNA genes of the yellow

pufferfish. There was also a study on genus Takifugu which used PCR methods to

amplify a 598-bp fragment of mitochondrial 16S rRNA gene (Song et al., 2001).

2.8 Socio-economic of Locals

Despite its toxicity, pufferfish are a known delicacy and expensive in Asian region

(Lin et al., 2002) especially in Japan and China. In Sarawak, the local people of Kg.

Manggut, Betong had been consuming the yellow pufferfish regularly (Nor Azman et al.,

2013). The preparation needs to be done by the experts since it contains tetrodotoxin

(TTX) that is lethal to the consumers. According to the local people, yellow pufferfish is

consumed and sold in the market located in Betong. These fishes are either sold fresh,

12

dried or salted. There was even a festival on yellow pufferfish held annually in August.The

local people stated that the event that are usually held during the festivals are; fishing

activities and selling of the fish and even preparing and cooking the yellow pufferfish.

13

3.0 Materials and Methods

3.1 Study site

The study site of this study is Batang Saribas of district Betong. The site is about

five hours away from Kuching, Sarawak. The selection was because of 1) accessible by

road and 2) report on the abundance of species by the local people and the previous

studies.

Figure 3.1 Sampling site, Batang Saribas, Sarawak (N 1°24’0”, E 111°31’0”)

14

3.2 Samples Collection

Four individuals of yellow pufferfish were collected from Batang Saribas with the

help of local people. The samples were then stored in cooler box with ice cubes while

being transported back to Aquatic Molecular Laboratory of Unimas. Photographs of the

samples were taken for record and also morphological assessment. In the laboratory, a

small portion of the samples’ tissue was taken and transferred into blank eppendorf tube

with 70% ethanol. The preserved samples and the rest of the fresh samples were then

stored in -20°C freezer for further analysis.

3.3 Morphological assessment

The samples were placed on a dark coloured background and photographed

individually. The morphometric characters namely total length (TL), standard length (SL)

was measured using a ruler in centimetre (cm) and recorded (Figure 3.3). Meristic

characters which are the numbers of anal fin rays and dorsal fin rays were also counted and

recorded.

Figure 3.2 Illustration of pufferfish showing total length(TL), standard length(SL), dorsal fin and anal fin (Santinni et al., 2013a)

SL

TL

Dorsal

fin

Anal fin

15

3.4 Total Genomic DNA Extraction

The modified CTAB protocol which was originally published by Doyle and Doyle

(1987) was used in the total genomic DNA extraction of pufferfish tissue. A small portion

of fish tissue was minced and transferred into a 1.5μl eppendorf tube, labeled as X1. A

volume of 200μl 1x CTAB buffer and 0.5μl of Proteinase K was transferred into the tube.

The tube was then vortexed by using Gilson® GVLaband incubated in water bath

(Wisebath, WISD) at 60°C for about an hour. The tube was occasionally inverted to mix

the solution inside the tube. Then, a total of 700μl of chloroform-isoamyl alcohol (CIA)

was added and the tube will be shaken gently by inverting the tube. Centrifugation was

done at 1300 rpm for 15 minutes at 4°C by using CF15RX HIMAC centrifuge machine.

Three layers were formed. A volume of 500μl of the uppermost layer was then transferred

into a new eppendorf tube, X2. After that, 500μl of absolute EtOH (100%) was added,

inverted and left at -20°C overnight. The tube X2 was centrifuged the next day at 1300 rpm

for 15 minutes at 4°C. The tube was taken out from the machine and the absolute EtOH

(100%) was poured out. A volume of 500μl of 70% EtOH was then added and tube X2 is

centrifuged again. The 70% EtOH was removed and the pellet was dried at room

temperature for an hour. The pellet was re-dissolved in 50µl deionized distilled water

(ddH2O) and stored at -20°C for gel electrophoresis.

3.5 Optical Density Reading

5µl of each DNA sample obtained was diluted with 495µl of ddH2O to obtain 100 x

dilutions. Then, the optical density reading of each sample was done by using ultraviolet

spectrophotometer, Ultrospec 1100 pro in Genetic Engineering Lab, FRST. The machine