universiti putra malaysiapsasir.upm.edu.my/id/eprint/40041/1/ib 2013 17r.pdfkeputusan untuk...

UNIVERSITI PUTRA MALAYSIA

MOHAMAD FAIZUL BIN MAT ISA

IB 2013 17

DEVELOPING OF PROTOCOL FOR EGG INCUBATION AND LARVAL CULTURE OF Tachypleus gigas Muller

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DEVELOPING OF PROTOCOL FOR EGG INCUBATION AND LARVAL

CULTURE OF Tachypleus gigas Muller

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfillment of the Requirements for the Degree of Master of Science

July 2013

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos, icons,

photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless

otherwise stated. Use may be made of any material contained within the thesis for non-

commercial purposes from the copyright holder. Commercial use of material may only be

made with the express, prior, written permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfilment of the requirement for the degree of Master of Science

DEVELOPING OF PROTOCOL FOR EGG INCUBATION AND LARVAL

CULTURE OF Tachypleus gigas Muller

By


July 2013

Chairman : Annie Christianus, PhD

Institute : Bioscience

Culture of horseshoe crab in laboratory for future restocking most likely is the

best solution so far in order to reduce the possibility of extinction of this species.

Establishment of suitable protocols is crucial to successfully hatch the eggs and

culture the larvae of Tachypleus gigas. Studies were carried out at MTDC

Laboratory, Putra Science Park, Universiti Putra Malaysia, Serdang, Selangor.

The objectives of this study were divided into two main parts. First, to determine

the effects of salinity, watering frequency and incubation medium on the hatching

of T. gigas eggs. Second, to determine the effects of salinity, temperature, culture

medium and stocking density on growth and survival of T. gigas larvae. Eggs,

larvae and sand were collected from three natural spawning sites in Sitiawan

(Perak), Banting (Selangor) and Muar (Johor), Malaysia. This research consists

of seven experimental studies. Three experiments were carried out on eggs and

four experiments on larvae. In the first, second and third experiment, effects of

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water salinities (15, 20, 25, and 30 ppt), watering frequencies (once in 1, 3 and 6

day/s), and different incubation medium (water and sand) on the incubated eggs

were investigated. As for the fourth, fifth, sixth and seventh experiment, effect of

salinities (15, 20, 25, 30 ppt), temperatures (ambient, 30 and 32˚C), using sand

and water medium, and stocking densities on horseshoe crab larvae was studied.

Results from experiment 1, showed that at the end of the incubation period,

watering with salinity of 25-30 ppt produced significantly larger eggs diameter

(P<0.05) while highest percentages of hatching at 30 ppt salinity. In experiment 2,

it was found that percentages of hatching were significantly higher (P<0.05) when

watered once a day and three days. As for experiment 3, at the end of the

incubation period, there was no significant different (P>0.05) in the eggs diameter

and percentage of hatching between sand and water medium.

Results for the fourth experiment (salinity) on instars 1 to 4 (1st to 6

th month) and

instars 4 to 7 (6th

to 12th month) only showed significant different (P<0.05) in

percentage of survival at 4th

instar stage while all parameters (prosomal width,

weight and survival) were significantly different (P<0.05) from 4 to 7th instar

stages. In the fifth experiment, percentage of survival was highest (P<0.05) at

30˚C. As for the sixth experiment, significant increments (P<0.05) were observed

for prosomal width, weight and survival when cultured in sand at the end of the 12

month period. In the seventh experiment, no significant different (P>0.05) for the

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prosomal width, weight and survival of T. gigas larvae were observed when

cultured at stocking densities of 20, 40 and 60 larvae/L.

Based on the findings of this study, it can be concluded that the most suitable

salinity and watering frequency for the incubation of horseshoe crab eggs are

between 25 to 30 ppt and once in 3 days, respectively. Both sand and water are

suitable media to incubate T. gigas eggs. As for the experiments on larvae, the

best salinity for optimum survival is between 20 to 30 ppt at temperature of 30˚C.

Meanwhile stocking density and culture media does not affect the growth and

survival of the larvae. Observation on the growth of T. gigas larvae throughout the

study period showed that prosomal width may not be a reliable indicator of

growth since the size will increase significantly whenever larvae molt. Therefore

it is better to measure the growth of T. gigas larvae through weight increment.

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Abstrak tesis dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan ijazah Master Sains

MEMBANGUNKAN PROTOKOL UNTUK PENGERAMAN TELUR DAN

KULTUR LARVA Tachypleus gigas Muller

Oleh


Julai 2013

Pengerusi : Annie Christianus, PhD

Institut : Biosains

Kultur belangkas dalam makmal untuk pelepasan stok pada masa akan datang

mungkin satu penyelesaian yang terbaik untuk mengelakkan kepupusan spesies

ini. Memperkenalkan protokol yang sesuai adalah penting untuk menetaskan telur

dan mengkultur larva Tachypleus gigas. Kajian dijalankan di Makmal MTDC,

Taman Sains Putra, Universiti Putra Malaysia, Serdang, Selangor. Objektif kajian

ini dibahagikan kepada dua bahagian utama. Pertama, untuk menentukan kesan

saliniti, frekuensi pemberian air dan medium untuk pengeraman telur ke atas

penetasan telur T. gigas. Kedua, untuk menentukan kesan saliniti, suhu, medium

kultur dan densiti stok ke atas tumbesaran dan kemandirian larva T. gigas. Telur,

larva dan pasir diambil dari tiga lokasi semulajadi pembiakan di Sitiawan (Perak),

Banting (Selangor) dan Muar (Johor), Malaysia. Kajian ini merangkumi tujuh

eksperimen. Tiga eksperimen dijalankan ke atas telur dan empat ke atas larva.

Dalam ekperimen pertama, kedua dan ketiga, kesan saliniti air (15, 20, 25, dan 30

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ppt), frekuensi pemberian air (sekali dalam 1, 3, dan 6 hari), dan medium

pengeraman berbeza (air dan pasir) ke atas telur dikaji. Untuk eksperimen

keempat, kelima, keenam dan ketujuh, kesan saliniti (15, 20, 25, dan 30 ppt), suhu

(ambien, 30, DAN 32˚C), penggunaan medium pasir dan air, dan densiti stok

larva belangkas dikaji

Keputusan eksperimen pertama menunjukkan bahawa pada akhir tempoh

pengeraman, pemberian air dengan saliniti 25 to 30 ppt menghasilkan diameter

telur yang lebih besar (P<0.05) dengan peratus penetasan yang paling tinggi

(P<0.05) pada saliniti 30 ppt. Dalam ekperimen kedua, didapati bahawa peratus

penetasan ketara lebih tinggi (P<0.05) bila pemberian air dijalankan sekali dalam

sehari dan tiga hari. Manakala untuk eksperimen ketiga, pada akhir tempoh

pengeraman, tidak terdapat perbezaan ketara (P>0.05) pada diameter telur dan

peratus penetasan apabila menggunakan medium pasir dan air.

Keputusan untuk eksperimen keempat (saliniti) keatas instars 1 hingga 4 (1

hingga 6 bulan) dan instars 4 hingga 7 (6 hingga 12 bulan) hanya menunjukkan

perbezaan ketara (P<0.05) dalam peratus kemandirian pada instar keempat,

manakala semua parameter (saiz prosoma, berat dan kemandirian) adalah ketara

berbeza (P<0.05) untuk peringkat instar ke 4 hingga 7. Dalam ekperimen kelima,

peratus kemandirian adalah paling tinggi (P<0.05) pada suhu 30˚C. Sementara

untuk eksperimen keenam peningkatan yang ketara (P<0.05) diperhatikan untuk

saiz prosoma, berat dan kemandirian apabila larva dikultur dalam pasir pada akhir

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tempoh 12 bulan. Dalam eksperimen ketujuh, tidak terdapat perbezaan yang

ketara (P>0.05) pada saiz prosoma, berat dan kemandirian larva T. gigas apablia

dikultur pada densiti stok 20, 40 dan 60 larva/L.

Berdasarkan hasil kajian ini, kesimpulan dapat dibuat bahawa saliniti dan

frekuensi pemberian air untuk pengeraman telur belangkas adalah di antara 25-30

ppt dan sehari sekali dan 3 hari sekali, setiap satunya. Kedua-dua pasir dan air

adalah sesuai digunakan untuk mengeramkan telur T. gigas. Sementara untuk

eksperimen ke atas larva, saliniti terbaik untuk kemandirian optima adalah di

antara 20 hingga 30 ppt pada suhu 30˚C. Sementara densiti stok dan kultur media

tidak memberi kesan ke atas tumbesaran larva. Pemerhatian ke atas tumbesaran

larva T. gigas dalam tempoh kajian menunjukkan bahawa saiz prosoma mungkin

tidak sesuai digunakan sebagai penunjuk tumbesaran kerana saiz akan meningkat

dengan ketara apabila larva bertukar kulit. Oleh itu, adalah lebih baik mengukur

tumbesaran larva T. gigas melalui peningkatan berat.

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ACKNOWLEDGEMENTS

First and foremost, I offer my sincerest gratitude to my supervisor, Dr. Annie

Christianus, for her guidance and critical review during my research. Her

perpetual energy and enthusiasm in research had motivated me to complete this

research. In addition, she was always accessible and willing to help, support and

guide me. I owe my deepest gratitude to my co-supervisors, Prof. Madya Dr. Che

Roos Saad and Dr. S.M. Nurul Amin for generating ideas of the whole research,

provide comments and help me in interpretation of results. In addition, I am also

grateful to the staff at Marine Science Laboratory (MARSLAB) and Aquaculture

Department, UPM, Mr. Muhammad Farhan Nazarudin, Mr. Zainan, and Mrs.

Nur Shafika Maulad Abd. Jalil for their kindness in lending their hand during the

research. I am indebted to many of my colleagues in Aquaculture Department,

Zaaim Zahari, Mohammad Faizal Mansor, Noor Fazielawanie Mohd Rashid, Eng

Hueh Theng, Md. Fairuz Ariffin Md. Yunos and Siti Fatimah Sulaiman for their

invaluable assistance, advices, support who inspired me in completing this study.

My deepest gratitude goose to my family especially my mother, Puan Hajjah

Azizah Abu Taat and my father, Tuan Haji Mat Isa Shafie, PJK, PPN for their

unflagging love and support throughout my life. A special thanks to my brothers,

Mohamad Ridza and Mohammad Imran for their understanding and unconditional

love. This dissertation is simply impossible without them. This research was

supported by Fundamental Research Grant Scheme (FGRS), project no:10201,

Vote no: 5524074. Lastly, sincere regards and blessings are extended to all who

supported me in one form or another during completion of this project.

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I certify that a Thesis Examination Committee has met on 23rd

July 2013 to

conduct the final examination of Mohamad Faizul Mat Isa on her thesis entitled

“Developing of Protocol for Egg Incubation and Larval Culture of Tachypleus

gigas (Muller, 1785)” in accordance with the Universities and University College

Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15

March 1998. The committee recommends that the student be awarded the Master

of Science.

Members of the Thesis Examination Committee were as follows:

Muta Harah Zakaria, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Hassan Mohd Daud, PhD

Associate Professor

Faculty of Veterinary Medicine


(Internal Examiner)

Hishammudin Omar, PhD

Senior Lecturer

Faculty of Science


(Internal Examiner)

Mazlan Abd Ghaffar, PhD

Professor

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

Malaysia

(External Examiner)

---------------------------------

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies


Date:

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This thesis was submitted to the senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Masters of Science.

The members of the Supervisory Committee were as follows:

Annie Christianus, PhD

Senior Lecture



(Chairman)

Che Roos Saad, PhD

Associate Professor



(Member)

S.M. Nurul Amin, PhD

Senior Lecture



(Member)

__________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations

which have been duly acknowledge. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra

Malaysia or at any institution.

_______________________________


Date: 23 July 2013

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TABLE OF CONTENTS

Page

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLE

LIST OF FIGURE

LIST OF ABBREVIATIONS

CHAPTER

1 INTRODUCTION

2 LITERATURE REVIEW

ii

v

viii

ix

xi

xv

xvii

xviii

1

4

2.1 Status horseshoe crab in Malaysia

2.2 Threat to horseshoe crab population

2.3 Taxonomy and morphology of horseshoe crab

2.4 Life cycle and reproduction of horseshoe crab

2.5 Eggs and larvae development

2.6 Molting and growth of horseshoe crab

2.7 Water quality

2.8 Biomedical and pharmaceutical applications

4

4

5

8

11

12

15

16

3 GENERAL MATERIALS AND METHOD

3.1 Location and sample collection

3.2 Eggs condition

3.3 Sand preparation

3.4 Seawater preparation

3.5 Sampling instruments

4 DEVELOPING OF OPTIMAL PROTOCOL FOR

INCUBATING Tachypleus gigas (Müller 1785) EGGS

4.1 Introduction

4.2 Materials and Method

4.2.1 Experimental animals

4.2.2 Preparation of sand and trays

4.2.3 Conditioning of T. gigas eggs

4.2.4 Experimental design

4.2.5 Data collection

4.3 Results and Discussion

4.3.1 Hatching and development of T.

gigas eggs incubated in sand at different

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salinities

4.3.2 Hatching of T. gigas eggs incubated in sand

and watered at different frequencies

4.3.3 Hatching and development of T.gigas eggs

incubated in sand and water media

4.3.4 Morphological changes during the embryonic

development

4.4 Conclusion

5 THE EFFECT OF SALINITIES, TEMPERATURE,

CULTURE MEDIA AND STOCKING DENSITY ON

GROWTH AND SURVIVAL OF Tachypleus Gigas

(Muller, 1785) LARVAE

5.1 Introduction

5.2 Meterials and Methods

5.2.1 Location of study

5.2.2 Eggs collection

5.2.3 Larvae maintenance

5.2.4 Feeding preparation

5.2.5 Experimental design

5.2.6 Experiment 4: Effect of salinity on the

growth and survival of first instar and fourth

instar T. gigas larvae

5.2.7 Experiment 5: Effect of temperature on the

growth and survival of first instar T. gigas

larvae

5.2.8 Experiment 6: Effect of culture media on

growth and survival of first and fourth instar T.

gigas larvae

5.2.9 Experiment 7: Effect of stocking density on the

growth and survival of first instar T.

gigas larvae.

5.2.10 Water quality determination

5.2.11 Data collection

5.2.12 Data analysis

5.3 Results and Discussion

5.3.1 Effect of salinities on growth and survival

of T. gigas larvae

5.3.2 Effect of temperature on growth and

survival of T. gigas larvae

5.3.3 Effect of culture media on growth and


5.3.4 Effect of stocking density on growth and


5.4 Conclusion

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6 GENERAL DISCUSSION, CONCLUSION AND

RECOMMENDATION

REFERENCES

APPENDICES

BIODATA OF STUDENT

PUBLICATIONS

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