Preliminary Study on Sergestid Shrimps (Acetes) of Sarawak Coastal Waters based

on Morphology and PCR-RAPD Approaches

Christine Anthonius (20821)

Bachelor of Science with Honours

(Aquatic Resource Science and Management)

2012

Faculty of Resource Science and Technology

Preliminary Study on Sergestid Shrimps (Acetes) of Sarawak Coastal Waters based on

Morphology and PCR-RAPD Approaches

Christine Anthonius (20821)

A project report submitted in partial fulfillment of the

Final Year Project (STF 3015) Course

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2012

DECLARATION

No portion of work referred in 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.

______________________________

Christine Anthonius

Aquatic Resource Science Management

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

I

ACKNOWLEDGEMENT

First of all, I wish to express my deepest gratitude to my supervisor, Assoc. Prof. Dr Othman

Bojo and my co-supervisor, Dr Ruhana Hassan. I would like to thank them for their dedication

and continuous constructive ideas, guidance, encouragement and assistance in the preparation

and completion of my Final Year Project.

I would also like to express my deepest thanks to all the Post-Graduate students including Ms.

Nurhartini Kamalia Yahya, Ms. Farah Adibah Esa, Ms. Nursyuhaida Md Shahid, Mr Mohd

Izwan Zulaini, Mr Mohd Khairulazman bin Sulaiman, and all the lab assistants who are willing

to help me in everything, continuous advice and encouragement especially during my project

schedule.

For my fellow friends, Siti Rokhaiya Haji Biollah, Nurul Jannah binti Ismail, Noor Liyana binti

Osman, Nur Hazwanie Izyan binti Mohd Nizam, Junirah Jamil and all Final Year students, I

would like to thank them for their moral support and cooperation in the lab work and outside the

lab schedule.

Last but not least, my deepest thanks goes to my beloved family and parents, Mr Anthonius

Yassin Kunan and Mdm Malader binti Padasian, for their moral support, motivation and endless

encouragement, from the beginning till the end.

II

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 4

2.1 Taxonomy of Acetes spp. 4

2.2 Morphology on Acetes spp. 5

2.3 Distribution of Acetes spp. 6

2.4 Importance of Acetes spp. 7

2.4.1 Ecological importance 7

2.4.2 Economic importance 8

2.5 Random Amplified Polymorphic DNA (RAPD) 9

2.6 Research on Acetes spp. 12

3.0 Materials and Methods 14

3.1 Samples collection 14

3.2 Laboratory work 15

3.2.1 Samples identification 15

3.2.2 Total genomic DNA extraction 16

3.2.3 Agarose gel electrophoresis 18

3.2.4 Gel documentation of DNA bands 19

3.2.5 Polymerase Chain Reaction (PCR) for RAPD 20

4.0 Results and Discussion 23

4.1 Morphological description and species identification 23

4.2 Total genomic DNA extractions 36

4.2.1 Agarose gel electrophoresis 36

4.3 PCR-RAPD 40

4.3.1 Comparisons of PCR-RAPD profiles 43

5.0 Conclusion and Recommendation 45

5.1 Conclusion

5.2 Recommendation

45

45

6.0 References 46

7.0 Appendix

III

List of Abbreviations

bp base pair

CTAB Cetyltrimethylammonium bromide

DNA Deoxyribonucleic acid

ddH2O Double distilled water

dNTP Deoxyribonucleotide triphosphate

EtBr Ethidium bromide

MgCl2 Magnesium chloride

NaCl Sodium chloride

PCR Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNA

rpm Revolutions per minute

sec Seconds

TBE Tris-borate-EDTA

V Volt

IV

List of Tables

Table Title Page

Table 3.2 PCR Master Mixture (modified from Aziz et al., 2010) 21

Table 3.3 Amplification program for PCR-RAPD of OPA07 primer

(adapted from Aziz et al., 2010)

21

Table 4.1 The morphology and measurement of an individual of

Acetes spp. with the mean and standard deviation of the

overall individuals of the respective sample (Appendix 1).

24

Table 4.2 The morphology and measurement of a single individual of

A. intermedius from Bako with the mean and standard

deviation of the overall individuals of the respective sample

(Appendix 2).

27

Table 4.3 The morphology and measurement of an individual A.

indicus from Bintulu with the mean and standard deviation

of the overall individuals of the respective sample

(Appendix 3).

31

Table 4.4 Comparison of mean and standard deviation for

morphological characteristics of sample A, B and C

35

Table 4.5 Comparisons of PCR-RAPD profiles between Acetes

shrimps

43

V

List of Figures

Figure Title Page

Figure 2.1 Taxonomic hierarchy of Acetes spp. (adapted from

Forest and Klein, 2004).

4

Figure 2.2 The body plan of a male Acetes (Omori, 1975). 6

Figure 3.1 Map showing the location of each samples obtained. 14

Figure 3.2 Flow chart of the modified CTAB DNA extraction

protocol (Doyle & Doyle, 1987).

17

Figure 3.3 PCR profile for RAPD using OPA07 primer on

Acetes shrimps.

22

Figure 4.1 Dried Acetes spp. purchased from Kuching market

measured in centimeters (cm).

23

Figure 4.2 A. intermedius from Bako measured in centimeters

(cm).

26

Figure 4.3 (a) The triangular apex of telson of A. intermedius in

50X magnification.

28

Figure 4.3 (b) A diagram of the triangular apex of telson of A.

intermedius.

28

Figure 4.4 (a) The procurved tooth between the bases of first

pleopods in 50X magnification of A. intermedius.

28

Figure 4.4 (b) A diagram of the procurved tooth of A. intermedius. 28

Figure 4.5 (a) Inner margin of basis of third legs without sharply

pointed or blunt projection in 50X magnification of

A. intermedius.

29

Figure 4.5 (b) A diagram of the blunt projection of A.intermedius.

29

Figure 4.6 (a) First segment of antennular peduncle at most as long

as second and third segments together observed in

50X magnification of A. intermedius.

29

Figure 4.6 (b) A diagram of the antennular peduncle of A.

intermedius.

29

Figure 4.7 A. indicus from Bintulu measured in centimeters

(cm).

30

Figure 4.8 (a) The triangular apex of telson of A. indicus in 50X

magnification.

32

Figure 4.8 (b) A diagram of the triangular apex of telson of A.

indicus.

32

Figure 4.9 (a) Procurved tooth present between bases of first

pleopods in A. indicus in 50X magnification.

32

Figure 4.9 (b) A diagram of the procurved tooth of A. indicus. 32

VI

Figure Title Page

Figure 5.0 (a) Inner margin of basis of third leg with sharply

pointed projection in 50X magnification of A.

indicus.

33

Figure 5.0 (b) A diagram of the inner margin of basis of third leg

with sharply pointed projection in A.indicus.

33

Figure 5.1 (a) The lower antennular flagellum with one clasping

spine in male A. indicus in 50X magnification.

33

Figure 5.1 (b) A diagram of the lower antennular flagellum with

one clasping spine in A.indicus.

33

Figure 5.2 (a) Petasma without pars astringens in male individual of

A. indicus observed in 50X magnification.

34

Figure 5.2 (b) A diagram of petasma in male individual of A.

indicus.

34

Figure 5.3 Agarose gel photograph showing DNA extraction

products of Acetes shrimp

36

Figure 5.4 (a) Agarose gel electrophoresis photograph showing the

PCR-RAPD multiple bands for sample B (Bako)

which was A. intermedius.

40

Figure 5.4 (b) A diagram of RAPD bands of A. intermedius from

Bako with OPA07 primer amplification.

40

Figure 5.5 (a) Agarose gel electrophoresis photograph showing the

PCR-RAPD multiple bands for A. indicus (Bintulu).

41

Figure 5.5 (b) A diagram of RAPD bands of A. indicus from

Bintulu with OPA07 primer amplification.

41

1

Preliminary Study on Sergestid Shrimps (Acetes) of Sarawak Coastal Waters based on

Morphology and PCR-RAPD Approaches

Christine Anthonius

Aquatic Resource Science and Management Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Acetes spp. is a type of Sergestid shrimp which is common in Malaysian coastal waters. It is highly gregarious

and is a seasonal occurring shrimp. Acetes are locally known as „udang geragau‟ in Peninsular Malaysia. In

Sarawak, the locals usually called them as „bubok‟. In Malaysia, this shrimp is made into various food products.

This species is also known for its prominent role in ocean food web. Lack of studies on Acetes shrimp inhabiting

Sarawak waters had limit the public to maximize the potential role of Acetes shrimp. Therefore, this study aims to

record the morphological details of Acetes that occurs in Sarawak coastal waters and carry out preliminary

molecular studies on Acetes spp. Based on the morphological characters, the Acetes samples from Bako and

Bintulu were identified as Acetes intermedius and Acetes indicus, respectively. The Acetes samples from Kuching

failed to be determined up to species level due to its dried state. PCR-RAPD profile from Bako and Bintulu

samples had been documented. The percentage of polymorphic bands for sample B is 71.43 %. On the other

hand, sample C (Bintulu) showed only two bands unique to the respective species. The percentage of

polymorphic bands of Bintulu sample was 50 %. There were two monomorphic bands observed which were 500

bp and 800 bp, respectively.

Key words: Acetes spp., morphology, CTAB, PCR-RAPD, polymorphic.

ABSTRAK

Acetes spp. adalah sejenis udang Sergestid yang biasa di perairan pantai Malaysia. Ia adalah berkelompok dan

merupakan udang yang hidup bermusim. Acetes secara tempatannya dikenali sebagai udang geragau di

Semenanjung Malaysia. Di Sarawak, penduduk tempatan biasanya memanggil ia sebagai bubok. Di Malaysia,

udang ini dihasilkan menjadi pelbagai jenis produk makanan. Spesies ini juga dikenali dengan peranan

utamanya dalam jaringan makanan di laut. Ketiadaan data untuk kajian molekular terhadap udang Acetes di

Kuching telah menghadkan orang awam untuk memaksimakan peranan udang Acetes yang berpotensi. Oleh itu,

kajian ini bertujuan untuk merekod ciri-ciri morfologi Acetes yang terdapat di perairan pantai Sarawak dan

menjalankan kajian molekular awal Acetes spp.. Berdasarkan karakter morfologi, sampel Acetes daripada Bako

dan Bintulu masing-masing dikenalpasti sebagai Acetes intermedius dan Acetes indicus. Sampel Acetes dari

Kuching gagal dikenalpasti pada peringkat spesies disebabkan keadaan yang kering. Profil PCR-RAPD dari

sampel Bako dan Bintulu telah didokumentasi. Peratus band polimorfik untuk sampel B adalah 71.43%.

Sebaliknya, sampel C dari Bintulu adalah 50%. Terdapat dua band monomorfik yang dilihat masing-masing iaitu

500 bp dan 800 bp.

Kata kunci: Acetes spp., morfologi, CTAB, PCR-RAPD, polimorfik.

2

1.0 Introduction

Acetes spp. is a type of shrimps that inhabit coastal and estuarine waters. Acetes are highly

gregarious (Xiao & Greenwood, 1993). They form an important component of coastal

zooplankton communities and play an important role in the dynamics of coastal ecosystems,

especially in lagoons, seagrass beds and mangrove swamps, by linking vegetal materials,

phytoplankton, zooplankton and animals at higher trophic levels (Xiao & Greenwood, 1993).

Apart from its small size, Acetes shrimps contribute as food for the growing human

population. Acetes range from 10-40 mm in total length and are widely distributed in the world

(Omori, 1975; Holthuis, 1980). In Malaysia, this shrimps are often made into shrimp paste or

also made into various kinds of food products. In Sarawak, Acetes spp. provides local

fishermen with extra income during its season. During certain parts of the year, they form

conspicuous aggregations near the shore and are fished mainly with push net and fixed bag net

that is set near the shore against the flow of the tide (Arshad et al., 2007).

There were some studies of Acetes in Malaysia for example; Aziz et al. (2010) had studied

about the population characterization of A. japonicus using RAPD technique. Other previous

studies include Oh et al. (2010) about the catch composition using Estuarine Set Bag Net

(ESBN) in coastal waters of Johor followed by the distribution records of A. intermedius in

both Bintulu and Malacca coastal waters (Amin et al., 2008; Arshad et al., 2007).

3

There is no record on Acetes spp. study in Kuching area. Moreover, molecular studies on

Sarawak Acetes shrimps are lacking.

The objectives of this study are:

i. To document the PCR-RAPD profiles of Acetes shrimps sampled from

Kuching, Bako, and Bintulu, Sarawak coastal waters.

ii. To record the morphological details and species of Acetes spp. that occurs

in Sarawak coastal water.

4

2.0 Literature Review

2.1 Taxonomy of Acetes spp.

Acetes are small and planktonic organisms that can be found living in the estuaries and

shallow coastal waters. These shrimps are common especially in the tropical and subtropical

regions. Acetes shrimps belong to the family Sergestidae. Within the family, there are two

genera which are Acetes H. Milne Edwards, 1830, and Sergestes H.Milne Edwards, 1830

(Holthuis, 1980). The taxonomy of Acetes spp. is shown as in Figure 2.1.

Figure 2.1: Taxonomic hierarchy of Acetes spp. (adapted from Forest and Klein, 2004)

Kingdom : Animalia

Phylum : Arthropoda

Superclass : Crustacea Pennant,1777

Class : Malacostraca Latrielle,1802

Subclass : Eumalacostraca Grobben, 1892

Superorder : Eucarida Calman, 1904

Order : Decapoda Latrielle, 1802

Superfamily : Sergestoidea Dana, 1852

Family : Sergestidae Dana, 1852

Genus : Acetes H. Milne Edwards, 1830

Species : Acetes americanus Ortmann, 1893

Acetes australis Colefax, 1940

Acetes chinensis Hansen, 1919

Acetes erythraeus Nobili, 1905

Acetes indicus H. Milne Edwards, 1830

Acetes intermedius Omori, 1975

Acetes japonicus Kishinouye, 1905

Acetes serrulatus (Krӧyer, 1859)

Acetes sibogae Hansen, 1919

Acetes vulgaris Hansen, 1919

5

2.2 Morphology of Acetes spp.

Acetes shrimps own the common malacostracan caridoid body-plan (Fig. 2.2). Their bodies are

elongated and compressed. They also have a well-built abdomen that is specialized for

swimming. Each somite is encircled with a dorsal tergum, a ventral sternum and two lateral

pleura. The carapace of Acetes has characteristic ridges and grooves. Besides that, Acetes has

shorter and smaller rostrum than that of penaeids. The hepatic and supraorbital spines are well-

developed while their compound eyes are stalked and mobile. The mouth of Acetes is ventral

and located between the mandibles. The anus is located at the ventral surface of the telson

towards its base (Xiao & Greenwood, 1993).

Based on the study by Arshad et al. (2007), for male Acetes, the lower antenular flagellum is

13 or 14 segmented with one clasping spine. The descriptive remarks also include the petasma

that agreed in bearing with pars astringens and capitulum of petasma with 4 subsequently large

hooks along the outer margin. The cox of the third pereiopod lacks a tooth on the distal inner

margin. Appendix masculine hold 3 hooks (Arshad et al., 2007).

On the other hand, the female lower antenular flagellum is 12 to 15 segmented. The first

segment of antennular peduncle at most as long as second and third segments combined. The

third pereiopod possess a small projection on the distal inner margin. There is a pair of distinct

protuberances on the anterior part of the third thoracic sternite; the sternite is concave in the

median part and is not produced backwards (Arshad et al., 2007). A small procurved tooth

exists at the bases of the first pereiopod in both sexes. The apex of the telson is triangle and

sharp-pointed. There is also a red spot on the proximal part of the endopods of the uropod.

Figure 2.2 shows the morphology of male Acetes.

6

Figure 2.2: The body plan of a male Acetes (Omori,1975) AM, appendix masculine; AF, antennal annular

peduncle; AS, antennal scale; CH, chela; CR, cornea; End, endopod; ES, eye stalk; Exp, exopod; GC, genital

coxa; HS, hepatic spine; LF, lower flagellum; Mxpd, maxiliped; PT, procurved tooth; Rostrum; RPS, red pigment

spots; UF, upper flagellum.

2.3 Distribution of Acetes sp.

According to Canoy (1980), ten of fourteen existing species in the genus Acetes are found in

the Indo-West Pacific and the Indo-Malay Archipelago region is particularly rich in species.

Epiplanktonic copepods occurring in the lower equatorial latitudes, i.e. species with their

breeding range restricted to lower latitudes between the equator and 20-30° north and south,

tend to show regional provincialisms (Canoy, 1980).

According to Canoy (1980), a similar tendency emerges from the distribution of Acetes where

northern and southern limits fall roughly between the 30° parallels. All species of Acetes are

restricted to Atlantic, Indo-West Pacific, or the eastern tropical Pacific Ocean. Moreover, the

species is absent from East Atlantic-Mediterranean or the islands of the Central Pacific

including Hawaii and New Zealand (Canoy, 1980).

7

2.4 Importance of Acetes spp.

2.4.1 Ecological importance

Acetes is basically a common neritic, epipelagic shrimp and it usually can be found in

estuaries and backwaters where fresh water from the land poses a major impact to the situation

(Canoy, 1980). This species also can withstand a great change of salinity. Other common

environmental features correlated with appearance of the species are:

1. the sea is shallow for a great distance from the shore;

2. the area is separated from the open ocean by a peninsula, submarine sills or numerous

islands;

3. the tidal range is considerable;

4. the bottom is covered with mud or sandy-mud

Acetes occurs in surface waters (0-5 m) in massive accumulation or swarms which is greatly

influenced by the tidal current, precipitation and winds (Jaiswar & Chakraborty, 2005). The

swimming activity of this shrimp is maximum during the night. Apart from its importance as

food resource for coastal fishery population, it also forms the most important food item of the

fishes along this coast (Jaiswar & Chakraborty, 2005). It is also known to play a prominent

role in the ocean food web.

8

2.4.2 Economic importance

According to Canoy (1980), Acetes certainly affords a major source of protein to some of the

people in the Indo-West Pacific. The most important species of the genus that contribute

greatly in zooplankton fishery includes A. chinensis, A. erythraeus, A. indicus, A. japonicus,

and A. vulgaris. Amin et al. (2007) stated that the Acetes shrimps are an important fisheries

commodity in Malaysia and have the potential for large-scale commercial aquaculture. Acetes

affords a major source of protein to some of the people in Asia and East Africa (Omori, 1975).

In 2004 the annual catch in west coast of Johor exceeded 1300 tons and constitutes 17.41% of

its total landing in Malaysia (Oh et al., 2010). In Asian countries, only a small proportion of

the catch is marketed as fresh shrimps; the greater proportion is dried, salted or fermented in

various ways for food (Amin et al., 2008). Acetes are converted into a variety of preparations

including „belacan‟, „petis udang‟, „sambal‟, „trassi udang‟ and „chincalok‟ (Oh et al., 2010).

Surplus shrimps are used in the preparation of animal or fish food, fed to poultry, or converted

into shrimps powder used as a fertilizer (Johnson, 1976).

9

2.5 Random Amplified Polymorphic DNA (RAPD)

During a search for polymorphism in a genome, if no precise DNA sequence is targeted, small

oligonucleotides (6 to 9 bases) of random sequence can be used that will fix on the target DNA

at random (Tagu & Moussard, 2006). This technique is known as Random Amplified

Polymorphic DNA (RAPD). RAPD is recognized for its ability in carrying out the initial

screenings in many loci and it can distinguish many different species of organisms

simultaneously (Lynch & Milligan, 1994).

Karim et al. (2010) reported that molecular markers have been proved as valuable tools in the

characterization and evaluation of genetic diversity inter- and intraspecies either within or

between populations. PCR-RAPD marker is a simple technique and it also does not require a

lot of cost or in other words inexpensive conduct (Bardacki, 2001). In addition, Amavet et al.

(2007) also stated that PCR-RAPD only require low quantities and medium quality of DNA to

detect the PCR-RAPD profile.

In this study, PCR-RAPD was used to generate the PCR-RAPD profile based on the

mentioned advantages. According to Black (1993), it allows a more random sample of genome

with rapid production of large amounts of genetic information (Zhivotovsky, 1999). In

addition, it allows scores of markers to be assayed on DNA extracted from a single organism

(Wilkerson et al., 1993).

Tagu and Moussard (2006) stated that unlike with conventional PCR targeted on an identified

sequence, a RAPD reaction uses a single type of primer and not a pair of different primers.

Based on a report by Williams et al. (1990), the standard RAPD technique utilizes short

synthetic oligonucleotides of random sequences which are about 10 bases long which act as

10

primers with a relatively small amount of total genomic DNA under low annealing

temperatures.

Wilkerson et al. (1993) also reported that for a RAPD band to be generated, the specific

primer needs to match a binding site of approximately 2 to 3 kilobase pairs of another binding

site which is oriented opposite to ensure the single oligonucleotide can prime replication in

each forward and reverse direction. The number and the size of amplified fragments depend on

length and sequence of short, single and arbitrary primers (Bardacki & Skibinski, 1994).

Amavet et al. (2007) stated that RAPD analysis is a multilocus technique detecting

polymorphism based on an amplification of random DNA segments. In a study by Arnold et

al. (1991), they reported that RAPD band may display a high degree of polymorphism besides

allowing the screening of multiple primers against taxa of interest. Additionally, their study

has proven to be a means of quickly identifying species-specific markers.

RAPD can be used to quickly obtain various fragments of genomic DNA that are analysed

(Tagu & Moussard, 2006). The presence or absence of the polymorphism is either caused by

nucleotide sequence divergence in primer sites or by insertions or deletions in the amplified

segment of template DNA (Amavet et al., 2007). This in turn will provide information of point

mutations by using RAPD primers.

Tagu and Moussard (2006) also stated that the amplification fragments permitting the location

of a polymorphism can be sub-cloned and sequenced to redefine a pair of primers that will

have a high probability of being specific to the analyzed sample and this is known as sequence

characterized amplified region or SCAR. RAPD bands are generally representative of

dominant genetic markers, which are inherited in a Mendelian fashion and can be used as

11

molecular diagnostic characters at different taxonomic levels (Williams et al., 1990). They

have been successfully applied for taxonomic identification and in population genetic surveys

(Hadrys et al., 1992).

RAPD analyses have been used in several extensive studies over the years and the researches

are still growing ever since. RAPD markers have been used to assess the genetic diversity of

medicinal plants, Tylophora rotundifolia (Sebastian et al., 2010) and genetic relationship of

saprophyte, Ganoderma spp. (Latifah et al., 2005). Besides that, this technique also been used

for other organisms especially fish species. In a study by Bardacki (2001), PCR-RAPD had

been successful in the determination of different types of tilapia species and sub-species.

Sameer et al. (2009) also used this technique to detect the genetic variation in the wild

populations of Indian major carps (IMCs). In addition, this technique was also used in typing

of bacterial strains, Listeria spp. (MacGowan et al., 1993) and molecular characterization of

bacterial strains, Salmonella enteriditis (Yaqoob et al., 2007).

Though RAPD markers have been useful in several studies, it also has limitation as reported

by Semagn et al. (2006) in which the majority of alleles segregate as dominant markers, and

hence the technique does not allow identifying dominant homozygotes from heterozygotes. In

addition, it was mentioned that the RAPD techniques produce fragments from homozygous

dominant or heterozygous alleles but no fragment is produced from homozygous recessive

alleles because amplification is disrupted in both alleles.

12

2.6 Research on Acetes spp.

Acetes sp. has gain many interest among researchers worldwide to search for more new

information about this tiny organism. Although there are various studies related to Acetes sp.,

but they are only restricted to reproduction and population dynamics, and the studies of larval

distribution (Oh & Jeong, 2003; Achuthankutty, 1979). Recently, considerable attention has

been paid to their role in the productivity of the oceans as well.

In Malaysia, the researches include the population characterization, reproductive seasonality

and maturation, the biology and population of the shrimps, and the distribution records of the

shrimp species in selected locations (Aziz et al., 2010; Amin, 2008; Amin et al., 2008; Arshad

et al., 2007). Omori (1975) has summarized the data available on the fishery of Acetes in

various countries.

In the previous years of studies, there are numerous papers that are related to the fishery of

Acetes of Peninsular Malaysia (Tham, 1950; Pathansali, 1966; Omori, 1975; Johnson, 1976 &

Ahamad, 1993). Most of them are concise except those of Omori (1975) and Ahamad (1993).

The researches were dedicated for taxonomic diagnosis. As a result, the molecular studies are

limited.

So far, there is only one molecular study by Aziz et al. (2010) which is the study of population

characterization of A. japonicus using the RAPD technique. The study was to assess the

genetic relationship between three populations of Acetes in Malaysia using RAPD marker

technique. The purpose of using RAPD is to provide information on the A. japonicus genetic

stock structure and to indicate the population differentiation between the A. japonicus from the

three main fishery regions of Peninsular Malaysia.

13

Based on the data available in GenBank (www.ncbi.com), there are only 11 nucleotide

sequences that belong to Acetes sp. (accession numbers AB583755.1 – EF467167.1) with only

5 for Acetes sp. and the remaining number for A. chinensis and A. erythraeus. Although there

are various elements that need further research about this shrimp, one should find the

advantages that could be obtained especially in the knowledge area.

14

3.0 Materials and Methods

3.1 Samples collection

For field sampling, the samples were obtained from Kuching, Bako, and Bintulu. The dried

Acetes (bubok kering) samples were brought from Kuching. The samples from Bako and

Bintulu were each obtained from the fishermen of respective locations.

Figure 3.1: Map showing the location of each samples obtained.

Bako

Kuching

Bintulu

MALAYSIA

Sarawak

15

3.2 Laboratory work

3.2.1 Samples identification

The samples collected from each locations were labeled as A, B, and C. The samples were

each from Kuching, Bako, and Bintulu, respectively. After that, the morphological works were

carried to ten individuals of shrimps from each sample. There were 30 individuals of shrimps

selected randomly. Next, the measurements of total length (TL), standard length (SL),

abdominal length (AL), carapace length (CL) and telson length (TLL) were recorded. The

measurements for each individual were carried out using thread and ruler which were observed

under the Motic SMZ-168 series stereo microscope. After the measurements were recorded,

the species identification of each samples were performed. This identification of species was

carried out according to the study by Omori (1975).