EXPLOITATION STATUS and FOOD PREFERENCE of ADULT TROPICAL HORSESHOE CRAB, Tachypleus Gigas

EXPLOITATION STATUS AND FOOD PREFERENCE OF ADULT TROPICAL HORSESHOE CRAB, Tachypleus gigas

MOHD RAZALI BIN MD RAZAK

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biosciences)

Kulliyyah of Science International Islamic University Malaysia

SEPT 2018

ABSTRACT

According to this study, local in Malacca more preferred to apply the modern method (fishing net) (65.85%) than traditional method (hand-harvest) (34.15%) to harvest the T. gigas from the wild (p<0.05), while locals in Pahang more preferred to apply traditional (56.1%) than modern method (43.9%). Frequency of the modern harvesting method application in Malacca (25 ± 10.48 times) was higher than the traditional method (2 ± 0.73 times) and also higher compared to the modern method application in Pahang (6 ± 3.45 times) (p<0.05). Quantity of harvested crabs per month for one individual was higher in Malacca (16,860 T. gigas) compared to Pahang (4,180 T. gigas). Foods conditions would substantially influence their edibility. However, horseshoe crabs might have specific behaviour to manipulate the edibility of the foraged food. A total of 30 males and 30 females were introduced with five different natural potential feeds, namely, gastropods (Turritella sp.), crustacean (Squilla sp.), fish (Lates calcarifer), bivalve (Meretrix meretrix) and polychaete (Nereis sp.). The conditions of introduced feeds had been manipulated based on the natural foods condition in nature; decayed and protected in shell, hardened outer skin and host-tubed. Female crabs took shorter response period (3.42 ± 2.42 min) toward surrounding food compared to males (13.14 ± 6.21 min). Horseshoe crabs showed preference toward unshelled gastropods. The preference percentage of female (50%) toward unshelled gastropods was higher than males (36.67%). There is no preference behaviour showed by T. gigas on the shelled, hardened outer skin and host-tubed feed. Only the female crabs showed rejection toward decayed food. Different population of horseshoe crabs might have different food preferences. Stomach contents analyses were conducted on 60 samples of male and female; 10 males and 10 females were trapped in fishing net during the incoming high tide and 20 males and 20 females were hand-harvested at the spawning beach (10 samples during pre-mating and 10 during post-mating). This study found that echinoderm served as a main food composition in the gut of males (50%) and females (51.94%) during the open sea migration phase. The main composition was substituted by macrophyte (males: 59.51% to 65.15%; females: 36.36% to 58.10%) as they arrived to the spawning area. Based on Electivity Index, male crabs showed positive preference toward polychaete (EI: 0.04) and macrophyte (EI: 0.19) at the spawning site while, the females showed positive preference toward bivalve (EI: 0.46). In this study, 30 males and 30 females were introduced with gastropod, crustacean and bivalve in different percentage (0.2%, 0.6%, 1.0%, 1.4%, 1.8%, and 2.2%) that expressed from horseshoe crabs body mass to determine the level of the satiation. After being harvested from the wild, male crabs did not significantly eat until two weeks in captivity while, females (51.43% ± 25.54) started to eat during the early first week. Two-way ANOVA study found that females T. gigas’ satiation levels (crustacean: 1.7% ± 0.08; bivalve: 1.8% ± 0.06; gastropods: 1.8% ± 0.04) were significantly higher than males (crustacean: 1.4% ± 0.02; bivalve: 1.6% ± 0.05; gastropods: 1.7% ± 0.08) for all feed types (F = 13.98, p<0.05). Gut transit times of males (crustacean: 17 ± 1.7 hrs; bivalve: 17 ± 1.7 hrs; gastropods: 23 ± 4.6 hrs) were significantly longer than females (crustacean: 10 hrs ± 1.7; bivalve: 12 hrs ± 0; gastropods: 19 hrs ± 1.7)(F = 3.72, p<0.05). Male crabs took shorter time (1 ± 0 hr) to achieve their satiation compared to female crabs (2 ± 0 hrs).

خالصة البحث ABSTRACT IN ARABIC

إْ اٌص١ادْٚ اٌّحٛ١ٍْ فٚ ٟال٠ت ِالوا، ٚفماً ٌٙزٖ اٌذساصت، ٠فضً اصخؼّاي اٌطش٠مت اٌحذ٠زت، أٞ اٌص١ذ باٌشبىت )ٍٝػ )٪98.;8 اٌطش٠مت ا١ٌذ٠ٚت اٌخم١ٍذ٠ت )67.48٪( الصط١اد صشطاْ حذٚة اٌحصاْ )Tachypleus gigas( ِٓ اٌبحش )p<0.05(، ب١ّٕا ٠فضً اٌص١ادْٚ اٌّحٛ١ٍْ فٚ ٟال٠ت با٘أغ اصخؼّاي اٌطش٠مت اٌخم١ٍذ٠ت )ٍٝػ )٪89.4 اٌطش٠مت اٌحذ٠زت )>.76٪(. واْ حىشاس اصخؼّاي طش٠مت اٌص١ذ اٌحذ٠زت فٟ ِالوا )58±;44.7 ِشة( أٍٝػ ِٓ اٌطش٠مت اٌخم١ٍذ٠ت )5±6:.4 ِشة( ٚوأج أٍٝػ وزٌه فٚ ٟال٠ت با٘أغ ِماسٔت باٌطش٠مت اٌحذ٠زت )9±6.78 ِشة( )p<0.05(. وأج و١ّت صشطأاث حذٚة اٌحصاْ اٌّصطادة شٙش٠ا ٌٍفشد اٌٛاحذ أٍٝػ فٚ ٟال٠ت ِالوا )94;،49 صشطأا( ِماسٔت بخٍه فٚ ٟال٠ت با٘أغ )4;7،4 صشطأا(. حؤرش اٌظشٚف اٌغزائ١ت بشىً وب١ش ٍٝػ صالح١ت اٌضشطأاث ٌالصخٙالن، ِٚغ رٌه لذ ٠ىْٛ ٌذٜ صشطاْ حذٚة اٌحصاْ صٍٛن ِحذد ٌٍخالػب فٟ ِذٜ صالح١ت اٌغزاء اٌّجّّغ. حُ حمذ٠ُ خّش أغز٠ت طب١ؼ١ت ِخخٍفت إٌٝ 64 ِٓ روٛس اٌضشطاْ 64ٚ ِٓ اإلٔاد. األغز٠ت وأج= بط١ٕاث األلذاَ ).ٚ ،)Turritella spاٌمشش٠اث ).ٚ ،)Squilla spاألصّان (ٚ ،(Lates calcariferرٚاث اٌصذفخ١ٓ (ٚ ،(Meretrix meretrixوز١شاث األشؼاس ).Nereis sp(. حُ حمذ٠ُ األغز٠ت اٌّمذِت ٍٝػ حضب طبؼ١ت األغز٠ت فٟ اٌطبؼ١ت؛ ِزً حٍه اٌّخؼفٕت، ٚاٌّصذّفت، ٚاٌضاوٕاث فٟ األٔاب١ب، ٚحٍه بجٍذ خاسجٟ لاس. أخزث إٔاد اٌضشطاْ فخشة اصخجابت ألصش )6.75±5.75 دل١مت( حجاٖ األغز٠ت اٌّح١طت ِماسٔت باٌزوٛس )46.47±9.54 دل١مت(. أظٙشث صشطأاث حذٚة اٌحصاْ حفضٍٙ١ا ٌبط١ٕاث األلذاَ ػذ٠ّت األصذاف، ح١ذ وأج ٔضبت حفض١ً اإلٔاد )84٪( ٌبط١ٕاث األلذاَ ػذ٠ّت األصذاف أٍٝػ ِٓ اٌزوٛس ):69.9٪(. ٌُ ٠ىٓ ٕ٘ان أٞ صٍٛن حفضٍٟ١ ِٓ صشطأاث حذٚة اٌحصاْ حجاٖ األغز٠ت اٌّصذّفت، ٚاٌضاوٕاث فٟ األٔاب١ب، ٚحٍه بجٍذ خاسجٟ لاس. سفضج إٔاد اٌضشطأاث فمط األغز٠ت اٌّخؼفٕت. ٌٚزٌه لذ ٠ىْٛ ٌٍّجػّٛاث اٌّخخٍفت ِٓ صشطأاث حذٚة اٌحصاْ حفض١الث غزائ١ت ِخخٍفت. أجش٠ج ححا١ًٌ ٌّحخ٠ٛاث اؼٌّذة ١ػ 94 ٍٝػٕت ِٓ اٌزوٛس ٚاإلٔاد، ح١ذ حُ ص١ذ 44 روٛس ٚ 44 إٔاد بشبان اٌص١ذ خالي اٌّذ اؼٌاٌٟ اٌّٛاجٗ، ٚحُ ص١ذ 54 ِٓ اٌزوٛس ٚ 54 ِٓ اإلٔاد فٟ شاطٝء ٚضغ اٌبٛ١ض ١ػ 44ٕاث خالي فخشة ِا لبً اٌخىارش ١ػ 44ٕٚاث أرٕاء اٌخىارش(. ٚجذث ٘زٖ اٌذساصت أْ شٛو١اث اٌجٍذ وأج اٌّىْٛ اٌغزائٟ اٌشئ١ضٟ فٟ أؼِاء اٌزوٛس )ٚ )٪84اإلٔاد )7>.84٪( خالي ِشحٍت اٌٙجشة فٟ اٌبحش اٌّفخٛح، ٚبؼذ٘ا أصبحج إٌباحاث اٌّائ١ت اٌّىْٛ اٌشئ١ضٟ )اٌزوٛس= 8.>8٪ إٚ ، ٪98.4 ٌٝاإلٔاد= 69.6٪ إػ )٪8;.44 ٌٕٝذ ٚصٛي اٌضشطأاث إٌٝ ِٕطمت ٚضغ اٌبٛ١ض. اصخٕاداً إٌٝ ِؤشش االخخ١اس، أظٙش اٌزوٛس حفض١ال ٚصٍٛوا إ٠جاب١ا حجاٖ ِخؼذداث األشٛان )ٚ )4.4<=EIإٌباحاث اٌّائ١ت )EI=4.79( فِٛ ٟلغ ٚضغ اٌبٛ١ض، فٟ ح١ٓ أظٙشث اإلٔاد حفض١ًٍا صٍٛوا إ٠جاب١ًا حجاٖ رٚاث اٌضذفخ١ٓ )EI=>4.4(. حُ فٟ ٘زٖ اٌذساصت حمذ٠ُ بط١ٕاث األلذاَ، ٚاٌمشش٠اث، ٚرٚاث اٌصذفخ١ٓ إٌٝ 64 ِٓ اٌزوٛس 64ٚ ِٓ اإلٔاد بٕضب ِخخٍفت )٪4.5، 5.5ٚ ،٪4.;ٚ ،٪4.7ٚ ،٪4.4ٚ ،٪4.9ٚ٪( اٌخٟ حُ اشخمالٙا ِٓ وخً أجضاَ صشطأاث حذٚة اٌحصاْ ٌخحذ٠ذ ِضخٜٛ اٌشبغ. بؼذ االصط١اد ِٓ اٌبحش، ٌُ ٠أوً روٛس اٌضشطاْ بشىً ٍِحٛظ حخٝ أصب١ػٛٓ فٟ اٌحجز، ب١ّٕا بذأث اإلٔاد )84.76٪ ± 58.87( بخٕاٚي اٌطؼاَ خالي اٌفخشة اٌّبىشة ِٓ األصبٛع األٚي. ٚجذث دساصت ANOVA اإلحصائ١ت راث االحجا١٘ٓ أْ ِضخ٠ٛاث شبغ إٔاد صشطأاث حذٚة اٌحصاْ )لشش٠اث= :.4٪ ± ;4.4؛ ٚرٚاث اٌصذفخ١ٓ= ;.4% ± 0.06؛ ٚبط١ٕاث األلذاَ= ;.4٪ ± 4.47( وأج أٍٝػ بشىً ٍِحٛظ ِٓ اٌزوٛس )لشش٠اث= 4.7٪ ± 4.45؛ ٚرٚاث اٌصذفخ١ٓ= 4.9% ± 4.48؛ ٚبط١ٕاث األلذاَ= :.4٪ ± ;4.4( ٌج١ّغ أٛٔاع األغز٠ت )p<0.05 ،13.98 =F(. أٚلاث ػبٛس اٌمٕاة اٌٙض١ّت ٌٍزوٛس )اٌمشش٠اث= :4 ± :.4 صاػت؛ رٚاث اٌمششح١ٓ= :4 ± :.4 صاػت ؛ بط١ٕاث األلذاَ= 56 ± 7.9 صاػت( وأج أطٛي بىز١ش ِٓ اإلٔاد )اٌمشش٠اث= 44 ± :.4 صاػت؛ رٚاث اٌصذفخ١ٓ= 45 ± 4 صاػت؛ بط١ٕاث األلذاَ >4 ± :.4 صاػت( )p<0.05 ،3.72 =F(، ٚاصخغشق اٌزوٛس ٚلخا ألصش )4 ± 4 صاػت( ٌخحم١ك شبٙؼا ِماسٔت باإلٔاد )5 ± 4 صاػت(.

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion; it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biosciences).

………………………………….. Zaleha binti Kassim Supervisor

………………………………….. Mohd Armi bin Abu Samah Co-Supervisor

………………………………….. Asnor Azrin bin Sabuti Co-Supervisor

I certify that I have read this study and that in my opinion; it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biosciences).

………………………………….. Mohammad Mustafizur Rahman Internal Examiner

………………………………….. Samsur bin Mohamad External Examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biosciences).

………………………………….. Mardiana binti Mohd Ashaari Head, Department of Biotecnology

This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biosciences).

………………………………….. Shafida binti Abd Hamid Dean, Kulliyyah of Science

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Mohd Razali Bin Md Razak

Signature ...... Date ......

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

EXPLOITATION STATUS AND FOOD PREFERENCE OF ADULT TROPICAL HORSESHOE CRAB, Tachypleus gigas

I declare that the copyright holders of this thesis are jointly owned by the student and IIUM.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Mohd Razali bin Md Razak

……..…………………….. ……………………….. Signature Date

ACKNOWLEDGEMENTS

In the Name of Allah, The Most Gracious and Most Merciful who has granted me the strength, patience and perseverance throughout this research. Without His Blessing and permission, this research work would not been possible to be completed.

I express my gratitude to my revered supervisors, Assoc. Prof. Dr. Zaleha binti Kassim (IIUM), Dr. Mohd Armi bin Abu Samah (IIUM) and Dr. Asnor Azrin bin Sabuti (IIUM) for their affectionate guidance and abiding encouragement to rectify and bolster my spirit along the course of this study.

I would like to address my acknowledgement to the directors of INOCEM Research Station, Cherok Paloh, Assoc. Prof. Dr. Zaleha Kassim (2015 – 2017) and Prof. Dr. Ahmed Jalal Khan Chowdhury (2017 – 2018) for her/his approval to use the invaluable aquaculture and laboratory facilities which really facilitating me along the study. Thank you to all International Islamic University Malaysia staffs for their guidance and help throughout this study.

My sincere thanks to Bro. Khairul and Bro. Habib (Science officer), Bro. Faizal, Bro. Hilmi and Bro. Zul (Assist. science officer) and Sis. Aisyah, Sis. Nisak and Sis, Wana (admin) for their highly valuable assistance in laboratory experiments, field sampling and instrument handling along this research work.

I gratefully acknowledge my indebtedness to The Ministry of Higher Education Malaysia for the assistance in providing fund for this study under the Fundamental Research Grant Scheme (FRGS 2015-2017), FRGS15-199-0440 and to Centre of Postgraduate Studies, IIUM in providing scholarships during my study period.

I must acknowledge my colleagues, Zulfadli bin Abd Nasser, Hazwani Hanim binti Hasnan and Ku Nazlee bin Ku Ahmad for their assistance to associate and tenure in ways more than one to achieve the objectives of this study.

I wish to record a deep sense of gratitude to my beloved father (Md Razak bin Abd Majid), mother (Saleha binti Ali) and siblings for their trust, good wishes and encouragement which always inspire my sagging spirit to go ahead.

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TABLE OF CONTENTS

Abstract ...... ii Abstract in Arabic ...... iii Approval Page ...... iiv Declaration ...... v Copyright Page ...... vi Acknowledgements ...... vii Table of Contents ...... viii List of Tables ...... xi List of Figures ...... xii

CHAPTER ONE: INTRODUCTION...... 1 1.1 Background Of The Study ...... 1 1.2 Statement Of The Problem ...... 5 1.3 Significance Of The Study...... 8 1.4 Research Objectives...... 10 1.5 Research Questions ...... 10

CHAPTER TWO: LITERATURE REVIEW ...... 11 2.1 Horseshoe Crabs Distribution ...... 11 2.2 Horseshoe Crabs Life Cycle ...... 13 2.3 Threats To Horseshoe Crabs Population ...... 14 2.4 Horseshoe Crabs Exploitation ...... 15 2.5 Horseshoe Crabs Spawning Ecology ...... 16 2.6 Horseshoe Crabs Feeding Ecology ...... 18 2.6.1 Horseshoe Crabs In Food Web ...... 18 2.6.2 Feeding Behaviours And Mechanisms ...... 21 2.7 Benthic Community ...... 23 2.8 Sea Urchin Population ...... 25

CHAPTER THREE: RESEARCH METHODOLOGY ...... 26 3.1 Fishery Aspect Of Horseshoe Crab In The Peninsular Malaysia: Exploitation Status ...... 26 3.1.1 Study Site ...... 26 3.1.2 Ethical Consideration ...... 27 3.1.3 Data Collection Method ...... 28 3.1.4 Data Analysis ...... 28 3.2 Feeding Mechanisms Of Adult Tropical Horseshoe Crab Toward Different Food Conditions ...... 29 3.2.1 Samples Collection ...... 29 3.2.2 Manipulation Of Feeds‟ Condition ...... 30 3.2.3 Horseshoe Crabs Behaviour And Acceptance Toward Feed Items Analysis ...... 31 3.2.4 Statistical Analysis ...... 32 3.3 Food Preference (Gut Content Analysis) Of Horseshoe Crabs ...... 33

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3.3.1 Samples Collection ...... 33 3.3.2 Gut Content Analysis ...... 35 3.3.3 Benthos Analysis...... 36 3.3.4 Data Analysis ...... 37 3.4 Measuring Asian Horseshoe Crabs Satiation (Food Intake), Gut Transit Time And Defecation Pattern ...... 38 3.4.1 Samples Collection ...... 38 3.4.2 Feed Consumption During Acclimation Period ...... 38 3.4.3 Satiation Test...... 39 3.4.4 Feed Consumption Period And Gut Transit Time ...... 41 3.4.5 Defecation Analysis ...... 41 3.5 Benthos Composition In Amuk-Amuk, Raja Muda And Air Leleh Coral Reefs Cherok Paloh, Pahang...... 42 3.5.1 Coral Reefs Location ...... 42 3.5.2 Tracing Coral Reef Location ...... 42 3.5.3 Benthos Collection And Analysis ...... 43

CHAPTER FOUR: DATA ANALYSIS AND PRESENTATION OF RESULTS ...... 44 4.1 Fishery Aspect Of Horseshoe Crab In The Peninsular Malaysia: Exploitation Status ...... 44 4.2 Feeding Mechanisms Of Adult Tropical Horseshoe Crab Toward Different Food Conditions ...... 46 4.2.1 Feeding Behaviours...... 46 4.3 Food Preference (Gut Content Analysis) Of Horseshoe Crabs ...... 53 4.3.1 Feeding Intensity ...... 53 4.3.2 Food Composition ...... 54 4.3.3 Food Selection...... 56 4.4 Measuring Asian Horseshoe Crab Satiation (Food Intake), Gut Transit Time And Defecation Pattern ...... 57 4.4.1 Horseshoe Crabs Acceptance Toward Feeds During Acclimation Phase ...... 57 4.4.2 Feeding Period, Satiation Level And Gut Transit Time ...... 58 4.4.3 Defecation Pattern ...... 60 4.5 Benthos Composition In Amuk-Amuk, Raja Muda And Air Leleh Coral Reefs Cherok Paloh, Pahang...... 61 4.5.1 Benthos Composition ...... 61

CHAPTER FIVE: DISCUSSION AND CONCLUSION ...... 63 5.1 Fishery Aspect of Horseshoe Crab in the Peninsular Malaysia: Exploitation Status ...... 63 5.2 Feeding Mechanisms Of Adult Tropical Horseshoe Crab Toward Different Food Conditions ...... 65 5.3 Food Preference (Gut Content Analysis) Of Horseshoe Crabs ...... 67 5.4 Satiation (Feed Ratio), Gut Transit Time And Defecation Pattern Of Horseshoe Crabs ...... 72 5.5 Conclusion ...... 75 5.6 Recommendations...... 76

REFERENCES ...... 78

APPENDIX A: PREPARATION OF FEEDING MECHANISMS TEST (FEEDING TRAY AND FEEDING TEST TANK DESIGN) ...... 93 APPENDIX B: MALE AND FEMALE CRAB’S RESPONSE PERIOD TOWARD FEEDS ...... 95 APPENDIX C: APPLICATION OF FISHING NET GEAR TO HARVEST THE WILD HORSESHOE CRABS ...... 96 APPENDIX D: HORSESHOE CRABS SPAWNING SITE SURVEY ...... 98 APPENDIX E: MALE AND FEMALE CRAB’S FOOD SELECTION ...... 102 APPENDIX F: HORSESHOE CRAB’S GUT ...... 103 APPENDIX G: MALE AND FEMALE CRAB’S ELECTIVITY INDEX (EI) ...... 105 APPENDIX H: MALE AND FEMALE CRAB’S GASTRO SOMATIC INDEX (GSI) ...... 106 APPENDIX I: PREPARATION OF HORSESHOE CRAB’S FEED FOR THE SATIATION TEST ...... 107 APPENDIX J: MALE AND FEMALE CRAB’S DEFECATION AMOUNT AND FREQUENCY PEARSON CORRELATIONS ANALYSIS (n: 0, 12, 24, 36, 48, 60, 72, 84 HRS) ...... 109 APPENDIX K: SEA URCHIN IN THE FISHERMAN FISHING TRAP ...... 110 APPENDIX L: CORAL REEF & SEA URCHIN SURVEY ...... 112 APPENDIX M: CONFERENCE AND PUBLICATIONS ...... 115

LIST OF TABLES

Table 3.1 The manipulation of the horseshoe crabs feeds to three conditions; fresh and protected, fresh and unprotected and decayed. 31

Table 3.2 The quantity of horseshoe crabs that used in the gut content analysis study that harvested during different spawning migration phases; open sea, pre-mating and post-mating. 34

Table 3.3 Coordinate and depth of Amuk-amuk, Raja Muda and Air Leleh coral reefs. 42

Table 4.1 Horseshoe crabs‟ (Mean ± SD) feeding duration. 47

Table 4.2 The behaviour of males T. gigas toward different food items and conditions. 49

Table 4.3 The behaviour of females T. gigas toward different food items and conditions. 50

Table 4.4 Percentage of feed consumption per hour 59

Table 4.5 Male and female crabs‟ satiation level and gut transit time. 59

Table 4.6 The defecation pattern of male and female T. gigas. 60

LIST OF FIGURES

Figure 2.1 Horseshoe crabs food web. 20

Figure 3.1 Locations of the conducted survey at Malacca and Pahang coastal areas. A: Sungai Duyung (N 02°10'28.0" E 102°17'22.2"); B: Anjung Batu (N 02°08'37.5" E 102°21'10.8"); C: Merlimau (N 02°07'44.6" E 102°24'54.3"); D: Balok (N 03°56'14.9" E 103°22'30.2"); E: Pantai Sepat (N 03°42'20.7" E 103°20'07.1"); F: Cherok Paloh (N 03°36'17.1" E 103°22'51.0") 27

Figure 3.2 A pair of amplexed Asian horseshoe crabs, Tachypleus gigas in Cherok Paloh (N 03°36'17.1" E 103°22'51.0") spawning beach during spawning season (May – July 2016). 30

Figure 3.3 The arrangement of feeding trays inside the feeding test aquarium (130×130×30 cm). Arrow = The movement of the feeding trays after each feeding test (clockwise). 32

Figure 3.4 Asian horseshoe crabs, Tachypleus gigas were collected by the fishing net method. 33

Figure 3.5 Locations of Cherok Paloh, Kuantan, Pahang. A: Beach, hand harvesting (03°36'27" N, 103°23'36" E). B: Open sea, netting (03°37'33"N 103°23'16"E). 34

Figure 3.6 Dissection track (black dashes line) on prosoma and opisthosoma ventral region of Tachypleus gigas. 36

Figure 3.7 Flow chart of the T. gigas satiation test. 40

Figure 4.1 Percentage of the local fishermen engaged with the hand harvest and netting methods in Pahang and Malacca, Malaysia 44

Figure 4.2 The quantity of the harvested horseshoe crab per catch per person (Mean ± SD) in Pahang and Malacca. Different small letters on the bars within the same study site indicate significant difference (p<0.01). Different capital letters on the bars within the same harvesting method indicate significant difference (p<0.01). 45

Figure 4.3 Frequency of the hand harvest and netting method application (Mean ± SD) in Pahang and Malacca per person per month 46

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Figure 4.4 Percentage of T. gigas’ acceptance toward different feed items and conditions. 47

Figure 4.5 A: T. gigas adducted the genital operculum. B: T. gigas abducted the genital operculum downward during eat to avoid the feed moves under the opisthosoma. C: Movement and position of the abducted genital operculum (side view). 51

Figure 4.6 Water flowing direction under the opisthosoma to riddance the suspended decayed flesh. 51

Figure 4.7 The movement of horseshoe crabs‟ telson. The telson was flexed to elevate the opisthosoma region. 52

Figure 4.8 A: Horseshoe crabs flexed joint between femur and patella to hold bivalve shell under the gnathobases. B: Pusher leg. C: Pedipalp. 52

Figure 4.9 The gut fullness (percentage ± SD) and gastro-somatic index (GSI ± SD) of male and female crabs that harvested at open sea and during pre and post spawning (beach). Different capital letters between spawning migration phases within same sex indicate the significant differences (p<0.05). 54

Figure 4.10 Photographs of food items found inside the gut of Tachypleus gigas; 55

Figure 4.11 The food composition (percentage) inside the gut of male and female horseshoe crabs, Tachypleus gigas. 56

Figure 4.12 The electivity indices (EI) of different major food items consumed by Tachypleus gigas. 57

Figure 4.13 Horseshoe crabs acceptance (percentage ± SD) toward introduced feeds during the acclimation phase. Different capital letters on bars within the same sex indicate the significant differences (p<0.05). 58

Figure 4.14 Male and female crabs‟ gut transit time and satiation level. 59

Figure 4.15 Defecation amount and frequency of horseshoe crabs. 60

Figure 4.16 A: Echinoderm (sea urchin). B: Gastropod. 61

Figure 4.17 Benthos composition in Amuk-amuk coral reef (3°41'51"N 103°24'13"E), Raja Muda coral reef (3°38'01"N 103°28'23"E) and Air Leleh coral reef (3°35'38"N 103°29'00"E) sediments. 62

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Figure 5.1 Locations of the sampling area and coral reefs. 70

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CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Horseshoe crabs are mysterious chelicerate that extant since 300 million years ago and have virtually unchanged for the past 150 million years (Rudkin and Young, 2009).

Their capability to adapt with the various environment changes have led them to remain in the marine ecosystem for million years. However, there are only four extant species left and have inhabited the realm sympatrically (Tan et al., 2009; Behera et al.,

2015). Atlantic species, Limulus polyphemus distribution is limited in the Atlantic region (Botton, 1984a, 1984b; Saunders et al., 1986; Nordstrom et al., 2006; Haramis et al., 2007; Jackson et al., 2007; Ehlinger and Tankersley, 2009; Brockmann and

Johnson, 2011; Rozihan and Ismail, 2012; Beekey et al., 2013; Niles et al., 2013;

Vasquez et al., 2015).

Meanwhile, Tachypleus gigas, Carcinoscorpius rotundicauda and Tachypleus tridentatus mainly have inhabited in the Asian region (Mikkelsen, 1988; Khan, 2003;

Lee and Morton, 2005; Christianus and Saad, 2007; Hu et al., 2009; Iwaoka and

Okayama, 2009; Tan et al., 2009) include Malaysia (Shakiba Zadeh et al., 2009;

Zaleha et al., 2010, 2012). It is worth mentioning that out of three Asian species, T. gigas, and C. rotundicauda could be found in the West and East Malaysian coastal waters while the distribution of T. tridentatus is only restricted to the East Malaysian coastal water (Sabah and Sarawak regions) as reported by Chatterji et al. (2008), Tan et al. (2009) and John et al. (2010). According to Manca et al. (2016), horseshoe crabs could be found in estuaries and surf-protected beach during non-monsoon (spawning season). Watson and Chabot (2010) study found that horseshoe crabs would stay

passively in the deep sea area by burrowing under the sand during monsoon (non- spawning season).

A few decades ago, horseshoe crabs were only utilised in agriculture activity as bio fertilizer and livestock feed due to low commercial value (Rudloe, 1982;

Kreamer and Michels, 2009). Horseshoe crabs in Malaysia are now gazetted as a fishery commodity under the jurisdiction of the Department of Fisheries (DoF). They are mainly known as food for local consumption or marketed to the neighbour country, Thailand (Chatterji et al., 2008; John et al., 2010). A farmer association in

Johor Bharu (Pertubuhan Peladang Kawasan Johor Bharu Selatan, PPKJBS) also recently has expended their economic activity in collecting horseshoe crabs for local food market (personal communication). Nowadays, horseshoe crabs have been emerged as an important resource in the medical world (Naqvi et al., 2004; R. A.

Fisher and D. L. Fisher, 2006; Gerhart, 2007).

Researchers have encountered the capability of the Amoebocyte Lysate in horseshoe crabs blue-blood to detect the present of bacteria endotoxin in drugs and pharmaceutical apparatus (Naqvi et al., 2004; Gerhart, 2007). However, the blood production is solely contributed by the Atlantic species, L. polyphemus. Recently,

Tachypleus gigas has been found to have capability to produce the Tachypleus

Amoebocyte Lysate (TAL) that has similar properties and could substitute our dependence on Limulus Amoebocyte Lysate (LAL) production (Gauvry, 2011).

Moreover, the number of the Atlantic horseshoe crab, L. polyphemus is dwindling

(Morton, 1999; Brockmann and Johnson, 2011; Chen et al., 2004; Davis et al., 2006;

Gauvry, 2011).

This intriguing discovery could elevate the commercial value of the Asian horseshoe crabs in future, since the market value of the blue copper-based blood is

worth multi-million dollar (Beekey et al., 2013). Nevertheless, surge in market value may serve as an intrinsic exploitation-depend mechanism that possibly lead to the overexploitation. Overexploitation may causes loss of matured crabs in the perturbed population as the consequent of entrapment in fishing nets in coastal waters

(Cartwright-Taylor et al., 2011) or hand harvesting activity at the spawning site, since mostly of the horseshoe crabs that migrating and landing in the spawning site during the spawning season are the matured crabs. These factors would increase the variation of density and maturity between horseshoe crabs‟ population that may cause further declination to the perturbed population and lead to the extinction problem.

In term of ecology, the present of the horseshoe crabs in the ecosystem are vital in associate to connect the energy transfer within coastal food web (Berkson,

2009). As the omnivorous benthic feeder (Carmichael et al., 2004), prey selection has been identified as horseshoe crab‟s behaviour during foraging activity (Botton, 1984a;

Chatterji et al., 1992). Horseshoe crabs‟ feed selection is tend to the benthic community namely; bivalve, polychaete, crustacean, gastropods, and macrophytes

(Botton et al., 2003). Walls et al. (2002) study identified several benthic species inside the gut of the Atlantic horseshoe crabs namely; mollusks including razor clam, macoma clam (Macoma spp.), blue mussel (Mytilus edulis) and worms such as polychaete and nemertean. However, intensive previous studies on feeding ecology of horseshoe crabs were mostly focus on L. polyphemus.

Study on feeding ecology of the Asian horseshoe crab, T. gigas by Chatterji et al. (1992) along the Balramgari beach at the Bay of Bengal found that molluscs species was the major food composition in the gut of T. gigas. However, horseshoe crabs‟ food preference might be depends on the availability and abundance of the feed between seasons and geological areas (Botton, 1984a; John et al., 2012a). A study on

the diet of mangrove horseshoe crabs, Carcinoscopius rotundicauda in Pahang by

John et al. (2012a) found that they prefer polychaetes over molluscs during the mating season.

Nowadays, the populations of horseshoe crabs in Malaysia are undergoing a rapid decline (Chatterji et al., 2008; Christianus et al., 2008; John et al., 2012b;

Robert et al., 2014). Adibah et al. (2015) stated that anthropogenic pressure is the main factor that leads to the depletion problem in horseshoe crabs population number.

Disturbance such as, habitat destruction (Itow et al., 1991), land reclamation

(Shinohara, 1989; Chen et al., 2004) and pollution (Itow, 1998) have decreased the number of the available nesting site, destroy their ecosystem and their natural preference feed as well. These consequences would decrease the number of the horseshoe crabs migrate into the perturbed spawning area during the spawning season.

Rozihan et al. (2013) study found that the philopatric characteristic of horseshoe crabs would cause breeding disability with another horseshoe crab population hence, lead to further extinction problem.

Preservation and conservation effort are needed to ensure this valuable organisms remain in the realm. However, studies on T. gigas preference feed and feeding behaviour are still scanty compared to L. polyphemus. Low commercial value from a few decades ago has led to the lack of extensive study (Kreamer and Michels,

2009; Adibah et al., 2015) and reliable data (Tan et al., 2009) on T. gigas preference feed. Information on horseshoe crab feeding mechanisms is needed to appraise the horseshoe crabs population preservation and conservation. The aims of this study are to determine the exploitation status of T. gigas and maturity of T. gigas population in west (Malacca) and east coast (Pahang) Malaysian peninsular, to identify T. gigas feeding behaviour toward different feeds conditions; protected in shell, hardened outer

skin and host-tubed and unprotected (freshly dead and decayed), to investigate T. gigas feeding ecology through gut content analysis and to measure T. gigas feeding behaviours in captivity; satiation level, feeding period, and defecation pattern.

1.2 STATEMENT OF THE PROBLEM

A few decades ago, horseshoe crabs were only utilised in agriculture activity as fertilizer and livestock feed due to low commercial value (Kreamer and Michels,

2009). At present in Malaysia, they are mainly known as food for local consumption or marketed to the neighbour country (Chatterji et al., 2008; Kassim et al., 2008; John et al., 2010). Recently, horseshoe crabs‟ blue-blood has emerged as an important raw material in the medical field (Naqvi et al., 2004; Gerhart, 2007). Malaysian horseshoe crabs, T. gigas has been encountered to have capability to produce Tachypleus

Amoebocyte Lysate (TAL) that could substitute the usage of Limulus Amoebocyte

Lysate (LAL) in detecting the present of bacteria endotoxin (Gauvry, 2011; John,

2012). These intriguing discoveries could increase the demand on Malaysian horseshoe crabs, T. gigas, thus increase their economic value and increased the harvesting activity at the spawning site.

Increase in exploitation activity may threat the population of the matured horseshoe crabs (Widener and Barlow, 1999; Shin et al., 2009; Mishra and Mishra,

2011). Since the greatest proportion of adult horseshoe crabs‟ mortality is due to human activity (Robert et al., 2014; Christianus et al., 2008), exploitation on horseshoe crabs should be monitored to ensure the sustainability of this resource.

However, most of the available information on horseshoe crabs exploitation was derived from the Atlantic species L. polyphemus. No reliable information on T. gigas exploitation between west and east coast of Malaysian peninsular. Different in

exploitation status also could cause variation in horseshoe crab maturity proportions in particular population, as the exploitation of horseshoe crabs is restricted to the matured females crabs (Manca et al., 2016).

Biological study has led to the increases of horseshoe crab rearing in the captivity by the researchers for research, education and biomedical applications

(Carmichael and Brush, 2012). Commonly, the wild harvested crabs priory will be retained in captivity for the acclimation process within two weeks before the subsequent analysis (Botton, 1984a; Hurton et al., 2005; Chabot et al., 2007; John,

2012; Coates et al., 2012; Smith et al., 2013) and will be further held in captivity for weeks along the experiments activity. During the holding phase, researchers require to feed the crabs and manage the defecation waste. However, there is no reliable information on the level of satiation, feeding period and defecation pattern of the

Asian horseshoe crabs, T, gigas during the holding period in captivity.

The anthropogenic pressures in T. gigas spawning site have led to the decreases in T. gigas population density (Shinohara, 1989; Itow, 1998; Chen et al.,

2004; Iwaoka and Okayama, 2009; Tanacredi et al., 2009; Adibah et al., 2015). As the crabs also conducting their foraging activity in the spawning site (Carmichael et al.,

2004), those pressures would intrinsically degrading the availability of their natural feeds. Previous study found that horseshoe crabs could change their feeding behaviour according to the environment changes. However, studies on T. gigas feeding behaviour and acceptances toward feeds‟ conditions are still unclear. Early study on horseshoe crabs feeding mechanisms has been described by a number of researchers that initially triggered by Lockwood (1870).

Those studies used to identify the function of the horseshoe crabs‟ appendages regarding to the feeding behaviour (Shuster, 1982). Pieces of food are initially

captured by the crab, using the chelate walking legs. These appendages, as well as the chelicerae, chilaria, and genital operculum, manipulate food to the gnathobases of the legs. However, the feeding behaviours were intensively studied on L. polyphemus.

There are several appendages descriptions in the previous studies are seems not similar compared to the Asian horseshoe crab, T. gigas since both of them are different in morphological appearance. The Asian horseshoe crabs might have their own feeding mechanisms in dealing with the surrounding feed.

In estuaries ecosystem, horseshoe crabs are known as the important predators that highly forage on benthic organisms (Carmichael et al., 2004). According to

Botton et al. (2003), horseshoe crabs natural feeds are restricted to benthic community such as, bivalve, polychaete, crustacean, gastropods and macrophyte. Those food types could be found in the intertidal zone. However, the information on T. gigas preference feed is still scarce compared to work on L. polyphemus. Preference feeds study on Asian species that conducted by John et al. (2012a) determined that horseshoe crabs in east coast of Malaysian peninsular preferred to consume polychaete. Nonetheless, the study was restricted to the mangrove species,

Carcinoscorpius rotundicauda.

Study on feeding ecology of the Asian horseshoe crab, T. gigas by Chatterji et al. (1992) along the Balramgari beach at the Bay of Bengal has found that molluscs species was the major food composition in the gut of T. gigas. However, this food preference might be depend on to the availability and abundance of the feed in the particular environment. John et al. (2012a) and Botton (1984a) studies found the food selections of horseshoe crabs were depend on the availability of the feed. As the population of the T. gigas species that distributed between Indian Ocean and South

China Sea separated by the Malaysian Peninsular barrier, their feeding ecology also

might be different. The differences in feeding ecology led Botton (1984b) and Botton and Haskin (1984) to conduct serial studies on the preference food of L. polyphemus that inhabit the New Jersey and Delaware beaches respectively during 1984. In addition, Carmichael et al. (2004) study on N and C stable isotopic in horseshoe crabs diet composition encountered a contrary result than the previous studies where, most of the food items inside the gut of horseshoe crabs were not came from the spawning ground.

1.3 SIGNIFICANCE OF THE STUDY

As discussed previously, commercial value and biotechnology study would increase the exploitation of horseshoe crabs from the wild. According to Manca et al. (2016), only the matured female horseshoe crabs will be harvested from the wild by the fishermen. Since the greatest proportion of adult horseshoe crabs‟ mortality is probably due to the exploitation activity (Daniels et al., 1998; Christianus et al., 2008;

Robert et al., 2014), it should be monitored to ensure the sustainability of this species.

As noted by Botton et al. (2015), the extensive study on T. gigas exploitation status in

Malaysian coast is urgently needed for further conservation. Horseshoe crabs‟ gut content analysis is crucial in order to identify their preference feed. Besides that, comparison between gut content of the horseshoe crabs that caught at different spawning migration phases could reveal their exact feeding time.

Combination between migration track and feeding time studies could be used to estimate their natural foraging ground. The ability of the horseshoe crabs to remain their existence since the past 150 million years has attracted the attention of ecologists and conventional biologist who believe that horseshoe crabs could altering their feeding behaviour and mechanism according to various environmental condition

(Zaleha et al., 2010; John et al., 2012b). Horseshoe crabs might have an ability to adapt with the changes of the feeds availability and manipulate the edibility of the foraged feed. Smith et al. (2013) study found that male horseshoe crabs would shift their preference feed to sea grass during the amplexus mating position.

Study on T. gigas feeding mechanisms toward different condition of feeds is needed to determine the capability of the Asian horseshoe crabs to manipulate the edibility of the surrounding food in nature and their preference feed. Thus, assessment on the suitability of food in the spawning site and the longevity of the spawning area to support the food requirement during spawning season can be conducted. These data are needed to appraise the preservation and conservation effort of T. gigas crabs‟ preference feeds and foraging ground (Robert et al., 2014). Feeding behaviour of horseshoe crabs in the captivity and nature are controlled by the endogenous circadian clock and it could be synchronized according to the surrounding environment (Chabot et al., 2008; Watson and Chabot, 2010).

There are a few horseshoe crabs behaviours namely; feeding period, satiation level and defecation pattern during the retention period in captivity that need to be detailed out. This information is needed in order to identify the behaviour of the crabs in captivity and evaluate the efficiency of the applied rearing method during the holding phase. Inadequate quantity of feed and poor waste management might lead to the nutrition deficiency and water deterioration problems respectively and subsequently affect the health of the crabs and decrease their performance. Horseshoe crabs are strongly requiring the habitats with higher water quality to maintain their health (Carmichael et al., 2004).

1.4 RESEARCH OBJECTIVES

The study aimed to achieve the following objectives:

i. To identify the exploitation status; the preference harvesting method, the

frequency of the preference harvesting method application per month and

quantity of the collected T. gigas per harvest in west coast (Malacca) and

east coast (Pahang) Malaysian peninsular.

ii. To determine T. gigas feeding behaviour toward different feed types and

conditions in laboratory trials.

iii. To identify and compare the stomach content and gastrosomatic index of

male and female crabs during different spawning migration phases; open

sea, pre-mating and post-mating period.

iv. To calculate the satiation level, feeding period and defecation pattern

(frequency and quantity) of Tachypleus gigas during the retention period

in the captivity.

v. To identify the possible foraging site of horseshoe crabs at the open sea

area by conducting benthos composition analysis.

1.5 RESEARCH QUESTIONS

i. How long the T. gigas takes to response, consume and digest the foraged

feeds?

ii. How the T. gigas reacts toward protected and decayed feeds?

iii. How much food T. gigas needs to achieve satiation?

iv. When and where the T. gigas conducts their foraging activity in the wild?

v. What is the most preference method by the local in Malacca and Pahang to

harvest the T. gigas?