STUDY OF MORPHOLOGY, STOMACH CONTENT AND TOXIN PROPERTIES OF PUFFER FISH IN LUNDU, SARAWAK. Nurfakhriah Binti Alias Bachelor of Science with Honors (Aquatic Resource Science and Management) 2013 Faculty of Resource Science and Technology
STUDY OF MORPHOLOGY, STOMACH CONTENT
AND TOXIN PROPERTIES OF PUFFER FISH IN
LUNDU, SARAWAK.
Nurfakhriah Binti Alias
Bachelor of Science with Honors
(Aquatic Resource Science and Management)
2013
Faculty of Resource Science and Technology
i
Acknowledgement
First of all, I would like to express gratitude to my supervisor Dr. Samsur Mohamad for the
useful comments and knowledge given throughout the learning process for this Final Year
Project. Besides, I would like to thank laboratory assistants Encik Nazri, Encik Zaidi and
Encik Zulkifli for the helps given during my laboratory session and also during the
sampling. Also, I want to thank my course mates, Nur Jamiatul Shaharum and Sabariah
Zulkfli for the helps during the sampling and also other course mates for the
encouragements. I also would like to thank my parents, who have supported me a lot
throughout this period.
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Table of Contents
Acknowledgement............................................................................................... I
Table of Contents ................................................................................................ II
List of Abbreviations .......................................................................................... III
List of Tables ….................................................................................................. IV
List of Figures …………………………………………………………………. V
Abstract …………………………………..……………………………………. 1
1.0 Introduction …….......................................................................................... 2
2.0 Literature Review .......................................................................................
2.1 Puffer Fish .............................................................................................
2.2 Distribution of Toxic Puffer Fish ...........................................................
2.3 Feeding Habit of Puffer Fish ..................................................................
2.4 Accumulation of Tetrodotoxin (TTX) ...................................................
2.5 Puffer Fish Poisoning .............................................................................
2.6 Methods Used in Detection of Tetrodotoxin (TTX)…………………...
2.6.1 High Performance Liquid Chromatography (HPLC)……………
2.6.2 Liquid Chromatography-Mass Spectrometry (LC-MS)………….
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5
7
8
9
10
11
11
12
3.0 Materials and Methods ................................................................................
3.1 Study Area ..............................................................................................
3.2 Sampling ................................................................................................
3.3 Species Identification .............................................................................
3.4 Physical Measurement ...........................................................................
3.5 Stomach Content Analysis ....................................................................
3.6 Toxin Extraction ....................................................................................
3.7 High Performance Liquid Chromatography ..........................................
3.8.1 Phosphate Buffer ..........................................................................
3.8.2 Sodium Buffer ...............................................................................
3.8.3 Oxidizing Reagent ........................................................................
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13
11
14
14
14
15
15
15
16
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4 Results and discussion...................................................................................
4.1 Species Identification of Puffer Fish Samples........................................
4.2 Morphological Assessment......................................................................
4.3 Physical Measurement.............................................................................
4.4 Meristic Count........................................................................................
4.5 Stomach Content Analysis ......................................................................
4.6 Toxicity Analysis ....................................................................................
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17
18
19
20
21
30
5 Conclusion .................................................................................................... 32
6 References .....................................................................................................
7 Appendices ………………………………………………………………...
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37
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List of Abbreviations
AcOH Acetic acid
AR Anal ray
BW Body weight
DR Dorsal ray
FR Fin ray
GPS Global positioning system
HL Head length
HPLC High Performance Liquid Chromatography
MU Mouse unit
NaOH Sodium hydroxide
PR Pectoral ray
SL Standard length
STX Saxitoxin
TL Total length
TTX Tetrodotoxin
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List of Tables
Tables
Description
Page
1 Taxonomic groups of spotted green and yellow puffer fish 17
2 The average of BW, TL, SL and HL of X. naritus 19
3 The average of BW, TL, SL and HL of T. nigroviridis 20
4 The range of AR, FR, DR and PR of X. naritus and T. nigroviridis 20
5 Prey items found in stomachs of T. nigroviridis 27
6 Prey items found in stomachs of X. naritus 28
7 Toxicity level of T. nigroviridis and X. naritus collected from Lundu 30
v
List of Figures
Figure
Description Page
1
Location of sampling area, Rambungan River and Sampadi Island
13
2
Leaves fragments 22
3 Prawn 22
4 Bivalves
23
5
Gastropod Species 1 23
6 Shell fragments of gastropod species 2
24
7 Oligochaete 24
8 Crab’s leg 25
9 Exoskeleton of prawn 25
10 Fish scales 26
11 Fish bones 26
1
Study of Morphology, Stomach Content and Toxin Properties of Puffer Fish in
Lundu, Sarawak
Nurfakhriah Alias
Aquatic Resource Science and Management Programme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Abstract
Morphological characteristics, stomach content and toxicity properties of puffer fish were studied. A sample
of 28 individuals of Tetraodon nigroviridis and 21 individuals of Xenopterus naritus were collected from
three sampling sites in Sarawak waters using three layered net. These two puffer fish species showed the
difference in their morphological characteristic (e.g., skin colour). Analysis showed that morphometric
measurement of T. nigroviridis collected from Rambungan River and Sampadi Island (BW: 47.04±11.60 g;
TL: 11.1±1.2 cm; SL: 8.0±0.7 cm; HL: 3.4± 1.3 cm) and (BW: 93.32±35.14 g; TL: 13.7±1.6 cm; SL:
10.5±1.2 cm; HL: 4.0±0.4 cm) respectively were different in their range of size. Analysis also showed the
difference in the range of size of X. naritus (BW: 335.00 ±35.36 g; TL: 23.3 ±2.8 cm; SL: 18.7 ±1.8 cm; HL:
4.8 ± 0.4 cm), (BW: 317.78±102.82 g: TL: 18.3 ±2.1cm; SL: 18.3 ±1.8 cm; HL: 5.5 ±0.7 cm) and
(BW:278.00 g; TL: 20.1 cm; SL: 16.3 cm; HL: 4.1 cm) collected from Rambungan River, Sampadi Island
and coastal area of Lundu respectively. Meristic count analysis of T. nigroviridis and X. naritus (AR: 11-14;
FR: 8-1I; DR: 13-15; PR: 20-23) and (AR: 24-28; FR: 10-12; DR: 30-35; PR: 16-18) respectively showed the
difference in the range of the rays counted. Stomach content analysis showed that T. nigroviridis was
carnivorous fish and X. naritus was omnivorous fish. The concentration of TTX detected in liver of X.
naritus was 39.39µg/g by using HPLC.
Key words: Tetraodon nigroviridis, Xenopterus naritus, morphological characteristics, morphometric
measurement, stomach content analysis, HPLC
Abstrak
Ciri-ciri morfologi, kandungan perut dan toksin bagi ikan buntal telah dikaji. Sebanyak 28 ekor Tetraodon
nigroviridis dan 21 ekor Xenopterus naritus telah ditangkap di tiga tempat persampelan di Sarawak
menggunakan pukat tiga lapis. Dua spesis buntal ini menunjukkan perbezaan dari segi ciri-ciri morfologi
(cth: warna kulit). Analisa menunjukkan perbezaan ukuran morfometrik T. nigroviridis antara Sungai
Rambungan dan Pulau Sampadi (BW: 47.04±11.60 g; TL: 11.1±1.2 cm; SL: 8.0±0.7 cm; HL: 3.4± 1.3 cm)
and (BW: 93.32±35.14 g; TL: 13.7±1.6 cm; SL: 10.5±1.2 cm; HL: 4.0±0.4 cm). Analisa juga menunjukkan
linkungan saiz X. naritus (BW: 335.00 ±35.36 g; TL: 23.3 ±2.8 cm; SL: 18.7 ±1.8 cm; HL: 4.8 ± 0.4 cm),
(BW: 317.78±102.82 g: TL: 18.3 ±2.1cm; SL: 18.3 ±1.8 cm; HL: 5.5 ±0.7 cm) and (BW:278.00 g; TL: 20.1
cm; SL: 16.3 cm; HL: 4.1 cm) yang ditangkap daripada Sungai Rambungan, Pulau Sampadi dan kawasan
pantai Lundu masing-masing menunjukan perbezaan. Analisa kiraan meristik T. nigrovirids dan X. naritus
AR: 11-14; FR: 8-1I; DR: 13-15; PR: 20-23) and (AR: 24-28; FR: 10-12; DR: 30-35; PR: 16-18) masing-
masing menunjukkan perbezaan dari segi lingkungan jari-jari sirip. Analisa kandungan perut menunjukkan
T. nigroviridis adalah ikan karnivor manakala X. naritus adalah ikan omnivor. Kepekatan TTX yang telah
dikenal pasti dalam hati X. naritus ialah 39.39µg/ menggunakan HPLC.
Kata kunci: Tetraodon nigroviridis, Xenopterus naritus, ciri-ciri morfometrik, ukuran morfometrik, analisa
kandungan perut, HPLC
2
Introduction
This study was conducted in Rambungan River and coastal area of Lundu including
coastal area of Sampadi Island, Sarawak. In previous year, Sarawak Forestry Corporation
(SFC) has issued a statement that Sampadi Island would be gazetted as marine protected
area (MPA) (The Star, 2012). According to Executive Order 13158, MPA is any means
area of the marine environment that has been reserved by Federal, State, territorial, tribal,
or local laws or regulations to provide lasting protection for part or all of the natural and
cultural resources therein. Sampadi Island is being considered to be included as one of
MPAs in Malaysia regarding to the high marine conservation value possessed by the
surrounding water bodies of the island.
Fishing is one of the main activities carried out by native communities living in
coastal area of Lundu. Puffer fish is one of target species by fishermen. The fish are caught
using traditional tools such as scoop net and modern equipments like trammel net and gill
net which are more efficient and commercially used by fishermen now days (Chuan &
Lim, 2004). The puffer fish caught will be sold in market and later are consumed by local
communities as one of their delicacy. A few poisoning cases had been reported in Sarawak
after consuming puffer fish. The poisoning is caused by tetrodotoxin (TTX) contained in
body tissues of the puffer fish. Despite this, little information is available about the TTX in
puffer fish species present in Sarawak waters.
Puffer fish is also recognized as blowfish and globefish (Sabrah et al., 2006). Puffer
fish is named so based on its ability to inflate its body where the inflation causes its
stomach to expand into peritoneal cavity surrounding the axial musculature. For example, a
3
marine puffer fish known as Diodon holocanthus will looks extremely spherical when
inflating its stomach.
A number of puffer fish species contain complex neurotoxin known as tetrodotoxin
(TTX). Improperly prepared puffer fish based meal has caused a series of food poisoning
related death cases in Southeast Asia (Chen et al., 2011). While in Malaysia, food
poisoning cases caused by ingestion of puffer fish are selectively reported. The puffer fish
poisoning are believed to be caused by TTX contained in the fish. TTX is highly potential
to disturb nervous system when entering human body system even at low concentration
(Arakawa, 2010). The puffer fish liver which is believed as the most toxic tissue in puffer
fish possesses a specific TTX uptake mechanism (Arakawa, 2010; Ikeda et al., 2010;
Matsumoto et al., 2007).
Local people of Sarawak including the native communities of Lundu area consume
puffer fish as one of their delicacies. Therefore, this study is important to assess the
biological data and to determine the toxin properties of puffer fish in the area in order to
provide public awareness about the neurotoxin contained in the puffer fish. In recent years,
only little data of biological and toxin properties have been documented for puffer fish in
Lundu coastal area. In previous study carried out by Muthukrishnan (2010), toxicity
analysis of puffer fish samples collected from Sampadi River was carried out using TLC.
The accuracy of toxin properties data of puffer fish in the river will be improved as during
this study, HPLC will be used for toxicity analysis instead of TLC. This is because HPLC
is able to provide more accurate data on toxin analysis compared to TLC. Other than type
of species, the location of where the samples are collected is another factor that influences
4
the ratio of toxicity level among the puffer fish samples (Ngy at al., 2008; Rodriguez et al.,
2012). Therefore, sampling will be conducted in two different locations; Sampadi Island
and Sampadi River to determine the difference of toxin properties of puffer fish between
these two locations.
The objectives of this study are:
1. To record data of puffer fish species present in Lundu District.
2. To analyze stomach content in order to determine the diet composition of puffer
fish population.
3. To determine the toxin properties of puffer fish in Lundu District using High
Performance Liquid Chromatography (HPLC).
5
2.0 Literature Review
2.1 Puffer Fish
Puffer fish belong to order Tetraodontiformers. ‘Tetra’ means four, ‘odontos’
means teeth and ‘formes’ means shape. Therefore, puffer fish species belong to this order
possess four teeth. Tetraodontiformers are divided into two families; family Diodontidae
and family Tetraodontidae. A total of 189 species and 28 genera of puffer fish belong to
family Tetraodontidae (Veeruraj, 2011). Puffer fish from family Diodontidae can be
identified due to spines on its body while the one from family Tetraodontidae possesses
smooth and sharpnose. Diodontidae can be distinguished from Tetraodontidae by the
presence of single tooth in each jaw of Diodontidae members.
Puffer fish inhabits marine water including tropical area and subtropical area of
Atlantic, Indian and Pacific Ocean, brackish water and freshwater area (Che Awin, 2008).
Most of puffer fish species live in tropical seas and they are normally found in shallow area
near shore waters (Mehmet, 2011) such as coral reef area. Puffer known as Lagocephalus
lunaris is a marine puffer fish species while puffer Tetraodon nigroviridis is a marine-
brackish species where this species can be found in river mouth area. For freshwater
environment, puffer species that can be found is T. leiurus.
According to Monaliza and Mohamad (2011), there are 12 species of marine-
brackish puffers have been identified in Sabah and Sarawak. Certain of the puffer fish
species do migration where they migrate into more brackish or freshwater habitats.
Common puffer fish species that can be found in Malaysian water are Lagocephalus
wheeleri, L. sceleratus and L. lunaris lunaris. While L. wheeleri, L. sceleratus and
6
Xenopterus naritus are puffer fish species that are consumed by a part of Malaysian
communities. Puffer fish, Lagocephalus sceleratus inhabits in ocean in the depths ranging
from 18 to 100 m and coral reef area (Mehmet, 2011).
Xenopterus naritus is widely present in Asian countries like China, Vietnam, Thailand,
Indonesia and Malaysia (Chuan & Lim, 2004). In Malaysian waters, the puffer fish
species is abundantly present in State of Sarawak only. X. naritus could be found living in
coastal water, estuaries and upper river area where the water salinity reaching zero. X.
naritus could be found in coastal water and mangroves area where the fish is abundantly
occur in estuaries of Batang Sadong, Batang Lupar and Batang Saribas in Sarawak.
According to toxicity study carried out by Monaliza and Mohamad (2011), X. naritus
is a non toxic puffer fish. According to Chuan & Lim (2004), the most promising period
for high catch for this species would be in March and October every year especially in the
middle of the months which king tide occurs and at the end of the months which springtide
occurs. The fish is a local delicacy where the flesh is directly cooking, salted or dried and
fermented. Other than flesh, the fish eggs also been dried. Tetraodon nigroviridis is widely
distributed in freshwater and brackishwater in tropical Asia from Sri Lanka to Indonesia,
northward to Vietnam (Matsunuma et al., 2011).
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2.2 Distribution of Toxic Puffer Fish
Marine puffer fish from family Tetraodontidae may contain highly toxic
neurotoxin, tetrodotoxin (TTX) (Ikeda et al., 2010; Islam et al., 2011; Ngy et al., 2008) or
saxitoxin (STX) (Nakashima et al., 2004) in their body tissues. In Tetraodontidae family
members, complex TTX is highly recorded in their livers and ovaries (Arakawa, 2010).
Tetraodontidae family members that are regarded as toxic puffer fish includes
Lagocephalus sceleratus (Rodriguez et al., 2011), Tetraodon nigroviridis, T.
steindachneri, and T. ocellatus (Ngy et al., 2008). L. lunaris, L. spadiceus and Arothron
stellatus found in Malaysian waters are toxic puffer fish that can cause food poisoning after
consumption (Monaliza & Mohamad, 2011). Toxicity analysis conducted on L. lunaris
shows that this species contain toxin in their muscle tissues (Rodriguez et al., 2011).
In recent years, STX has been discovered in several marine and freshwater puffer
fish species along with TTX (Abbott et al., 2009). Thus, certain puffer fish species may
possess TTX as their major toxin while STX as their minor toxin and vice versa.
According to Mehmet (2011), TTX can be found in liver, gonads, intestines and skin
tissues of puffer fish and is highly potential to cause the individuals that consume the
tissues to death.
For Japanese marine puffer fish recognized as Takifugu pardalis, T. poecilonotus
and T. vermicularis, STX is their minor toxin while in puffer known as Arothron
firmamentum, STX is its major toxin where the toxin is mainly accumulated in its ovary,
but its skin only contain TTX (Nakashima et al., 2004). Study carried out by Noordin
(2011) recorded that Carinotetraodon salivator contain low toxin level in its tissues.
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2.3 Feeding Habit of Puffer Fish
Studies conducted by Mohamad and Isa (2012) recorded that diet composition of
puffer fish is mainly composed of crustaceans, therefore puffer fish is regarded as
carnivorous fish. Other than crustaceans, puffer fish communities also consume mollusks
and small bivalves (Abbot et al., 2009). The bivalves are believed to accumulate toxin in
their muscles after been exposed to toxin-containing plankton such as Alexandrium
(Nakashima et al., 2004). According to study carried out by Mehmet in 2011,
Legocephalus sceleratus is a carnivorous puffer fish species where the puffer fish species
fed on shrimps, fishes, crabs, squids and cuttlefish. In other words, L. sceleratus feeds on
cephalopods, crustaceans and fishes.
Ikeda et al. (2010) suggest that toxin accumulation in puffer fish is originated from
their food chains. Matsumoto et al. (2010) also proposed that TTX accumulation in puffer
fish occur via bioaccumulation since cultured marine puffer fish are found not toxic
whereas non-toxic puffer fish turn into toxic after been fed on artificial TTX contained
diets during culture period (Matsumoto et al., 2010). The toxin is possibly absorbed
through the intestine and later distributed into tissues in various part of the puffer fish body
through blood circulation (Matsumoto et al., 2007).
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2.4 Accumulation of Tetrodotoxin (TTX)
TTX is predominantly accumulated in liver and ovary of puffer fish that caused
these organs to possess high toxicity level (Ikeda et al., 2010; Matsumoto et al., 2007;
Nakashima et al., 2004; Ngy et al., 2008), however, the toxin levels are vary among
different puffer fish species (Islam et al., 2011). A patient will suffer the most severe
poisoning when he or she ingests puffer fish liver (Rodriguez et al., 2012). Thus, the liver
can be regarded as the most toxic organ in puffer fish.
During maturation period, the rate of TTX-binding plasma activity increase and
TTX is transported from liver to ovary (Ikeda et al., 2010). As a result, TTX level increase.
TTX is transferred into skin tissues through circulatory system (Arakawa et al., 2010).
Muscle also contain toxin but in much lower level (Ikeda et al., 2010; Rodriguez et al.,
2011). In female puffer fish, toxicity level is high in liver during ordinary period and high
in ovary in maturation period (Ikeda et al., 2010). This demonstrated that TTX originated
from food chains is transported mainly into liver and skin tissues of puffer fish during
ordinary period while the toxin is predominantly accumulated in their ovary during
maturation period.
The toxin contained in puffer fish tissues that is mainly composed of TTX is
believed to be used by the puffer fish communities not only to protect themselves but also
their eggs from predators. This is because puffer fish communities possess the highest TTX
content during spawning season where their gonads size also increase during the spawning
season (Yu & Yu, 2002). Puffer fish called L. sceleratus scores the highest toxicity level
during spawning season. Meanwhile, Yu et al. (2004) suggested the toxin level in puffer
10
fish largely depends on TTX-producing bacteria living symbiotically in the host bodies.
However, there is critism on the ability of the bacteria in producing TTX.
According to Matsumoto et al. (2007) the accumulation of TTX in puffer fish liver
involves a specific mechanism known as carrier-mediated transport system where the
endogenous and exogenous compounds will be accumulated in liver. The accumulation
and excretion of TTX and STX involve binding protein (Nakashima et al, 2004). The
binding protein are known as puffer fish STX and TTX-binding protein (PSTBP) that play
critical role in plasma protein binding of TTX and STX in marine puffer fish from
Tetraodontidae family (Matsumoto et al., 2010).
2.5 Puffer Fish Poisoning
Food poisoning cases due to ingestion of toxic puffer fish has been widely reported
in coastal countries of East and South-East Asia, including in Japan, China, Taiwan (Islam
et al., 2011), Bangladesh (Islam et al., 2011; Rodriguez et al., 2011), Thailand (Rodriguez
et al., 2011) and Cambodia (Ngy et al., 2008) while in Malaysia puffer fish poisoning are
selectively occur. Limb weakness and progressive muscular paralysis are among the
symptoms exhibited by individuals suffering puffer fish poisoning and the poisoning may
lead them to death resulting from respiratory paralysis (Islam et al., 2011).
According to O’Leary et al., (2004), the poisoning resulted from the consumption
of the puffer fish is caused by the tetrodotoxin (TTX) contained in the puffer fish tissue
and TTX poisoning is an important issue in South-eastern Asia. Tetrodotoxin (TTX) is
heat-stable, water-soluble and a non-protein organic compound which is aminoperhy-
11
droquinazoline. The quinazoline derivative is regarded as one of the strongest paralytic
toxins today and TTX is named after the order from which puffer fish normally grouped
into, Tetraodontiformes (Mehmet, 2011).
According to Matsumoto et al. (2007), TTX is able to block voltage-gated sodium
channel after entering organism bodies where the individuals will suffer severe food
poisoning, leading to fatality. The ability of TTX as a blocker is associated with its
characteristic of high affinity to the sodium channels, and the blocking action will inhibits
depolarization and propagation of action potential, paralysing the peripheral nervous
system (Islam et al., 2011). Thus, TTX is a disturbance in sodium conductance and
neuronal transmission in skeletal muscle.
2.6 Methods used in detection of tetrodotoxin (TTX)
Methods that can be used in TTX detection include High Performance Liquid
Chromatography (HPLC) and Liquid chromatography–mass spectrometry (LC-MS).
2.6.1 High Performance Liquid Chromatography (HPLC)
HPLC is a quantitative method to detect tetrodotoxins (TTXs) in samples including
gastropods and puffer fish (Chen & Chou, 1998). According to O’ Leary et al. (2004),
HPLC system comprises of pump with a manual injector, analytical column and
fluorescence detector. The detector is set with an excitation wavelength lex of 380 nm, and
an emission wavelength lem of 505 nm. The fluorescent detection is followed by post-
column degradation. Phosphate buffer which is composed of heptasulfonic acid acts as
12
mobile phase in the system which is pumped through the HPLC column at 0.3 ml/min.
Data is collected using software such as Maxima Data collection software.
Meanwhile, sodium hydroxide (NaOH) solution is pumped at 0.3 ml/min through a
second HPLC pump into a mixing device. Then, the solution will be mixed with the mobile
phase eluant. This mix then passed through Teflon tubing which was immersed in a water
bath maintained at 95 8C. After passing through the Teflon tubing, the mix then entered
the fluorescence detector.
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3.0 Materials and Methods
3.1 Study Area
Figure 1 Location of sampling area, Rambungan River and Sampadi Island
3.2 Sampling
Puffer fish samples were randomly collected from Rambungan River (N01° 39.78’
E110° 07.54’) and coastal area of Sampadi Island (N01° 42.0’ E110° 03.50’) and coastal
water of Lundu, Sarawak using three layered net with mesh size of 4 cm for its inner layer
and 14 cm for its outer layer. The samples collected were kept in cooler box filled with ice
before being dissected. The puffer fish were dissected and transported back to laboratory
for further analysis. In laboratory, the samples were kept in freezer at temperature of -
20°C.
14
3.3 Species Identification
The morphological characteristic including colour and shape of their bodies,
numbers of tooth and spines distribution were observed using naked eyes. The distributions
of spots or stripes on their bodies were described. Species of the samples were identified.
3.4 Physical Measurement
Total length (TL), standard length (SL) and head length (HL) for each puffer fish
was measured to nearest 0.1 centimetre using measuring board (Simon & Mazlan, 2008).
TL of the samples was measured from tip of the snout to the tip of caudal fin. SL of the
samples was measured from anterior tip of the snout to the posterior end until the last
fleshy part excluding the caudal peduncle in a straight line. HL was measured from tip of
the snout to the posterior edge of the opercular bone. The puffer fish sampled were sorted
into specific range of size. The body weight (BW) of each puffer fish samples was
recorded using electronic balance to nearest 0.01 gram (Mehmet, 2011). The number of
anal fin, caudal fin, dorsal fin and pectoral fin of each sample were counted. The data were
recorded in a field sample data label sheet prepared earlier.
3.5 Stomach Content Analysis
Stomach content analysis of the puffer fish samples were carried out according to
methods used by Hyslop (1985). The stomachs were removed from the fish bodies and
placed in specimen bottles. The stomachs were preserved using 10% formalin to avoid any
further digestion activity by digestive enzyme contained in the stomachs. The preservation
also can prevent the contents of the stomach from decomposed. Further examination of the
stomach contents were carried out in laboratory. In laboratory, the stomachs were
15
transferred into 0.45 µm sieve and rinsed with running tap water. The items found in
stomach of the samples were observed under stereo microscope. The items examined were
identified.
3.6 Toxin Extraction
The puffer fish samples were dissected into gonad, liver, muscle and skin.
Approximately 5 grams each of gonad, liver and muscle were minced. Each sample was
mixed in same volume of 0.1% acetic acid (AcOH). Then, the mixture was heated in
boiling water bath at 100 ºC for 10 minutes. The slurry was centrifuged at 10 000 g for 15
minutes with 22 ºC using centrifuge KUBOTA 6200. Supernatant was collected from the
sample by filtration through 0.45µm milipore filter paper. Finally, the supernatant was
transfer into vial bottle and kept in freezer at temperature of 20 ºC.
3.7 High Performance Liquid Chromatography (HPLC)
HPLC was conducted using Waters 600 Controller HPLC system using a
Symmetry® C18 5µM (4.6 × 150 mm) column for TTX analysis.
3.7.1 Phosphate Buffer
In preparation of 60 Mm ammonium phosphate (Ph 5) buffer solution, 300 ml
Mili-Q water and 205 µ l phosphoric acid were mixed. 2M ammonium was added into the
solution to adjust the solution to pH 5. Approximately 1.0 g heptanesulfonic acid (HSA)
was added into the mixture followed by addition of Mili-Q water till the volume reach 500
ml. Then, 10 ml of acetonitrile was added. Finally, the mixture was filtered using 0.45 µm
Milipore filter paper.
16
3.7.2 Sodium Buffer
In preparation of 4N sodium hydroxide (NaOH) buffer solution, approximately
74.32 g of solid form sodium was dissolved in 500 ml Mili-Q water.
3.7.3 Oxidizing Reagent
. 60 mM ammonium phosphate (Ph 5), an ironpairing reagent was used as mobile
phase at flow rate 0.8 mL/min. The eluent from the column was mixed with an equal
volume of 4 N NaOH. Then, the mixture was heated in a reaction coil at 110ºC. The toxin
was detected by using a fluorescence detector (Waters 2475 Multi Fluorescense Detector)
at 505 nm emission with 381 nm excitation. The concentration of TTX was calculated
using formula below:
(a/b) × 5
Where, a = peak area of sample
b = peak area of TTX standard
The amount of TTX calculated was in unit of MU/g for 10 µl mixture injected into
HPLC system.
17
4.0 Results and Discussion
A total of 49 puffer fish specimens belong to family tetraodontidae comprising of
28 individuals of Tetraodon nigroviridis and 21 individuals of Xenopterus naritus were
sampled for this particular study. All collected specimens were analyzed in stomach
content analysis.
4.1 Species Identification
A total of 21 individuals of Xenopterus naritus were identified and this species is
also known as yellow puffer fish. X. naritus was identified according its prominent
yellowish colouration especially towards the lower part of the body (Chuan & Lim, 2004).
The remaining 28 samples collected were identified as Tetraodon nigroviridis or also
known as green-spotted puffer fish. The identification of T. nigroviridis was made
according to identification key feature by Mohsin and Ambak (1983) where tetaodontidae
fish that have dorsal fin with less than 20 rays and possesses black spots belong to genus
Tetraodon.
Table 1: Taxonomic groups of spotted green and yellow puffer fish
Common Name Classification
Spotted green puffer fish Order: Tetraodontiformes
Family: Tetraodontidae
Genus: Tetraodon
Species: Tetraodon nigroviridis
Yellow puffer fish Order: Tetraodontiformes
Family: Tetraodontidae
Genus: Xenopterus
Species: Xenopterus naritus
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Both Xenopterus naritus and Tetraodon nigroviridis belong to same order and
family which are tetraodontiformes and tetraodontidae respectively as shown in Table 1.
Common name of T. nigroviridis is spotted green puffer fish while X. naritus is known as
yellow puffer fish. These two species are believed to be given name based on their
morphological characteristic where T. nigroviridis has spots on its body and the body is
mainly covered with green colour skin while the skin of X. naritus is mainly yellow in
colour.
4.2 Morphological Assessment
X. naritus possesses yellowish skin colour especially towards the lower part of its
body. T. nigroviridis possesses yellow-green skin colour of upper part of its body while
off-white to grey underneath. Besides, T. nigroviridis possesses small dark brown spots on
upper part of its body. T. nigroviridis has rounded body in cross section. The head and
body is covered with spinules. The back and side of its body has dark greenish yellow with
many black spots while its body is white in colour. Its dorsa, anal and pectoral fins are pale
while its caudal fin is pale with several transverse dark lines.
Tetraododon nigroviridis has about similar morphological feature with
Tetraododon fluviatilis. Therefore, it is quite difficult to differentiate between these two
species. However, T. fluviatilis could be differentiated with T. nigroviridis by the presence
of one black spot which is relatively about the size of the eye, surrounded by a white area
behind the pectoral fin. According to key feature given by Mohsin and Ambak (1983), T.
fluviatilis has black caudal fin with white edges. However, T. nigroviridis has pale caudal
fin with dark lines.