FATTY ACID CONTENT in TWO NUDIBRANCH SPECIES Phyllidia Varicosa and Phyllidiella Pustulosa from EAST COAST PENINSULAR MALAYSIA

FATTY ACID CONTENT IN TWO NUDIBRANCH SPECIES Phyllidia varicosa AND Phyllidiella pustulosa FROM EAST COAST PENINSULAR MALAYSIA

ALI ABDALLAH ALQUDAH

A thesis submitted in fulfillment of the requirement for the degree of Doctor of Philosophy in Biotechnology

Kulliyyah of Science International Islamic University Malaysia

MARCH 2017

ABSTRACT

The present study aimed to explore the molecular phylogeny, fatty acid composition, food preferences, and feeding ecology of the selected nudibranch species Phyllidia varicosa and Phyllidiella pustulosa from two locations: the coastal waters of Balok, Pahang and Bidong Island, Terengganu, Malaysia. A 16S rDNA marker was utilized to identify the studied species to the species level. 16S rDNA sequences of each studied species, P. varicosa and P. pustulosa, were aligned and applied to the examination of the phylogenetic relationships among them. The divergence of sequences was adequate to identify the samples to the species level with the assistance of the GenBank database and the BLAST tool. The studied species from the coastal water of Balok and Bidong Island are the same species and there is no possibility of new species arising. Analysis of the fatty acid composition of P. varicosa and P. pustulosa were analysed by gas chromatography-mass spectrometry (GC-MS), it was found that 15:0, 16:0, 17:0, 18:0, 18:1(n-9), 20:1(n-9) and 20:4(n-6) were predominant. Based on the univariate analysis, there was no significant difference in total saturated fatty acid (SFA). In contrast, there was a significant difference between species and site in total polyunsaturated fatty acids (PUFA) p < 0.05. Besides that, the univariate analysis of odd-chain branched fatty acids (OBFA) was significantly different between the species but not the site, and the total percentages of OBFA in Balok samples were noted to be higher than the sum of OBFA in Bidong Island samples. There was a significant difference between species but no significant differences in site and interaction in total of monounsaturated fatty acids (MUFA). Based on the pairwise analysis, the fatty acid composition between the same species but different sites was also significantly different p < 0.05. The overall fatty acid composition was significantly different. Statistical analyses were conducted in Primer V6 + PERMANOVA using Euclidean distance similarity matrices, and the data were visualized using PCO plot and showed the main fatty acids contributed to the main differences. Analysis of the fatty acid composition of the sponge prey was also carried out in order to assess the predator-prey relationships between P. varicosa species and its sponge prey. In both sponge species, the main saturated fatty acids were 16:0 and 18:0. The positional isomers of 18:0 were the most common in both species with 6.78% in Xestospongia vansoesti and 5.3% in Aaptos suberitoides. Two demospongic acids 5,9-25:2 and 5,9 -26:2 were detected. The characterization of 5,9 - 25:2 was achieved based on the MS spectra to confirm the presence of demospongic acids in the nudibranch tissue and sponge species. As a conclusion, based on the fatty acid analysis and food preferences, the variation of nudibranch size between two different locations might come from different diet specificities.

خالصة البحث

هتدف ىذه الدراسة إىل إستكشاف البيئة الغذائية و النسل اجلزيئي ومقارنة األمحاض الدىنية والتفضيالت الغذائية لنوعني من الدود البزاق ومها Phyllidia varicosa و Phyllidiella pustulosa يف املياه الساحلية ملنطقة بالوء يف والية هبانج وجزيرة بيدونغ يف والية ترينغانو. إستخدم جني 61س من احلمض النووي الرايبوزي للتعرف على العينات املدروسة إىل مستوى النوع. مت ربط سالسل احلمض النووي 61 س للنوعني P. varicosa و .P pustulosa, وتطبيقها لدراسة عالقات النشوء والتطور فيما بينها. كان االختالف يف السالسل كايف للتعرف على العينات إىل مستوى النوع وذلك مبساعدة قاعدة بيانات بنك اجلينات وأداة ال BLAST. كانت االنواع املدروسة مثطابقة من كال املنطقتني وال يوجدإحتمالية لنشوء نوع جديد. مت دراسة حتليل تكوين األمحاض الدىنية الثنني من الرخويات الدودة البزاق . مت حتليل حمتوى األمحاض الدىنية لالنواع اليت مت اختيارىا بإستخدام الغاز الكتل الطيفي )GC-MS(, وكانت االمحاض الدىنية C18: 1 ,C18: 0 ,C17: 0 ,C16: 0 ,C15: 0 )ن9-(, C20: 1 )ن-9( وC20: 4 )ن- 1( لتكون االكثر تواجدا ً. إستنادا إىل التحليل اإلحصتئي إحادي املتغري, مل يكن ىناك اختالف كبري يف جمموع األمحاض الدىنية املشبعة )SFA(. يف املقابل, كان ىناك فرق كبري بني األنواع واملوقع يف جمموع األمحاض الدىنية املتعددة غري املشبعة )بوفا( p < 0.05. باإلضافة إىل ذلك, فإن التحليل أحادي املتغري سلسلة األمحاض الدىنية متفرعة و الفردية )OBFA( ختتلف اختالفا كبريا بني األنواع ولكن ليس يف املوقع, وكما لوحظ أن جمموع نسب OBFA يف عينات بلوء أعلى من جمموع OBFA يف عينات جزيرة بيدونغ . كان ىناك فرق كبري بني األنواع ولكن عدم وجودفروق كبرية يف املوقع والتفاعل يف جمموعو من األمحاض الدىنية غري املشبعة االحادية )MUFA(. إستناداً إىل حتليل اإلحصائي الزوجي, كان حمتوى األمحاض الدىنية بني نفس النوع يف املواقع أيضا خمتلفة إىل حد كبري p < 0.05. كان حمتوى األمحاض الدىنية بشكل عام خيتلف إختالفا كبرياً. . مت إجراء التحليل اإلحصائي يف V6 + PERMANOVA بإستخدام مصفوفات تشابو املساحة اإلقليدية وصورت بإستخدام رسم PCO والذي أظهر االمحاض الدىنية اليت تسببت يف إحداث إالختالف. أجري حتليل لتكوين األمحاض الدىنية لفريسة اإلسفنج أيضا من أجل تقييم العالقات بني املفرتسات والفرائس لنوع P. varicosa وفريسة اإلسفنج هلا. يف كال نوعي االسفنج, كانت األمحاض الدىنية املشبعة الرئيسية C18: 0 ,C16: 0. وكانت األيزومرات املوضعية حلمض 6:81 األكثر شيوعا يف كل األنواع مبقدار :..1٪ يف Xestospongia vansoesti و 3.5٪ يف Aaptos suberitoides. مت الكشف عن د وجواالمحاض الدىنية االسفنجية وىي 25:2- 5,9 و 26:2- 5,9. مت وصف احلامض الدىين 25:2- 5,9 والتحقق منو على أساس مقياس األطياف لتأكيد وجود األمحاض demospongiae يف نسيج الدودة البزاقة واإلسفنج أيضا. يف النهاية, إستنادا ً إىل حتليل األمحاض الدىنية والتفضيالت الغذائية , إختالف حجم الدودة البزاقة بني منطقتني رمبا أتى من إختالف النظام الغذائي.

APPROVAL PAGE

The thesis of Ali Abdallah Alqudah has been approved by the following:

______Shahbudin Bin Saad Supervisor

______

Muhammad Nor Bin Omar Internal Examiner

______Zainudin Bachok External Examiner

______Aileen Tan Shau Hwai External Examiner

______Md Yousuf Ali Chairman

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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.

Ali Abdallah Alqudah

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

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

FATTY ACID CONTENT IN TWO NUDIBRANCH SPECIES Phyllidia varicosa AND Phyllidiella pustulosa FROM EAST COAST PENINSULAR MALAYSIA

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 Ali Abdallah Alqudah

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

ACKNOWLEDGMENTS

First of all praises to Almighty Allah (SWT) for His blessing Who granted me good health and allowed me to complete this research during specified period of time.

I would like to express my endless thanks and deep gratitude to my supervisor, Assoc. Prof. Dr. Shahbudin Saad. Without his guidance and persistent help, this thesis would not have been possible. I am also grateful to my co-supervisors Assoc. Prof. Dr. Deny Susanti and Asst. Prof. Dr. Noor Faizul for their valuable time, guidance and encouragement throughout the course of this research.

I would like to express my eternal appreciation towards my parents (Abdallah Faleh Alqudah and Husniiyh Abd Almuhdy) who have always been there for me with all support and patience. Thank you for being so understanding and supportive. I wish to record a deep sense of gratitude to my brothers Abo Sultan, Abo Saif, Ahmmad and sisters for their good wishes and encouragement which always inspire me to go ahead.

I wish to express my deepest gratitude and utmost appreciation to my beloved wife, Reema Alqudah for her never-ending motivation and love, which sustained me through the endeavour of this work. My heart felt regard goes to my father in law, mother in law, sister in law and brother n law for their love and moral support.

I am thankful to my dear friends Br. Hamizan, Br. Husaini, Br. Fikri, Sr. Najwa Husna for their valuable assistance during my research. Not forgetting all of my friends who always cheer me up and help me during the hard times, especially Usaf Ali, Anas Albazirgan, Hosam Tamimi, Syed Mahmood, Mohammed Aljarousha, Hassen Sheik and Izzat Qaralleh. It has been great to know all of you during my time at IIUM. I wish to thank all academic and non- academic staffs of Kulliyyah of science whose involvement and contributions led to successful completion of my degree.

TABLE OF CONTENTS

Abstract ...... i Abstract in Arabic ...... ii Approval Page ...... iii Declaration ...... iv Copyright Page ...... v Acknowledgements ...... vi List of Tables ...... x List of Figures ...... xi List of Symbols ...... xiv List of Abbreviations ...... xv

CHAPTER ONE: GENERAL INTRODUCTION ...... 1 1.1 Background of the Study ...... 1 1.2 Problem Statement ...... 4 1.3 Research Hypothesis ...... 5 1.4 Objectives of the Study ...... 5 1.5 Significance of the Study ...... 6 1.6 General Research Methodology ...... 7 1.7 Thesis Organization ...... 11

CHAPTER TWO: LITERATURE REVIEW ...... 14 2.1 Taxonomy, Biology and Ecology of the Phyllidiid Nudibranchs ...... 14 2.1.1 Distribution of Phyllidiid Nudibranch Species ...... 15 2.1.2 Feeding Ecology of Phyllidiid Nudibranchs ...... 16 2.1.3 Description of Phyllidia varicosa and Phyllidiella pustulosa...... 17 2.2 Phylogeny of nudibranchs ...... 20 2.3 Using of mitochondrial DNA in phylogeny ...... 21 2.4 Mitochondrial 16s rDNA as a genetic marker ...... 22 2.5 Recent Studies on Phyllidiid Nudibranch ...... 23 2.6 Fatty Acids in Marine Organisms ...... 24 2.6.1 Molluscan Fatty Acids ...... 26 2.6.2 Fatty Acid Composition of Nudibranchs ...... 27 2.7 Fatty Acids as Biological Markers ...... 29 2.8 Sponge - Feeding Nudibranchs ...... 31 2.8.1 Fatty Acid Composition in Sponge - Feeding Nudibranchs ...... 32 2.9 Fatty Acid Nomenclature ...... 34 2.10 Overview of Three Lipid Extraction Methods and FAME Preperaion ...... 37 2.11 Secondary Metabolites from Marine Sponge - Feeding Nudibranchs ...... 41

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CHAPTER THREE: IDENTIFICATION AND PHYLOGENETIC INFERENCE IN DIFFERENT NUDIBRANCH SPECIES VIA MITOCHONDRIAL 16S RDNA ...... 44 3.1 Introduction...... 44 3.2 Material and Methods ...... 46 3.2.1 Sample Collection ...... 46 3.2.2 DNA Extraction ...... 47 3.2.3 Gel Imaging of Genomic DNA by Gel Electrophoresis ...... 48 3.2.4 DNA Quantification ...... 49 3.2.5 Polymerase Chain Reaction (PCR) ...... 49 3.2.6 PCR Cycling Conditions ...... 49 3.2.7 Gel Imaging and Sequencing ...... 49 3.2.8 Bioinformatics ...... 50 3.3 Results ...... 50 3.3.1 Genomic DNA Extraction ...... 50 3.3.2 DNA Quantification ...... 51 3.3.3 DNA Amplification by Polymerase Chain Reaction (PCR) ...... 51 3.3.4 BLAST search ...... 52 3.3.5 Phylogenetic Trees of Phyllidiid Nudibranchs ...... 54 3.3.6 Phylogenetic Analysis ...... 55 3.4 Discussion ...... 58 3.5 Conclusion ...... 60

CHAPTER FOUR: COMPARISON OF FATTY ACID COMPOSITION OF TWO NUDIBRANCH SPECIES Phyllidia varicosa AND Phyllidiella pustulosa ...... 61 4.1 Introduction...... 61 4.2 Materials and Methods ...... 63 4.2.1 Station Location and Description ...... 63 4.2.2 Sample Collection ...... 64 4.2.3 Body Size Measurement ...... 65 4.2.4 Lipid Extraction ...... 67 4.2.5 FAME ...... 68 4.2.6 Gas ChromatographyMass Spectrometry Conditions ...... 69 4.3 Statistical Analysis...... 69 4.3.1 Statistical Analysis and Data Configuration ...... 70 4.4 Results ...... 72 4.5 Discussion ...... 83 4.6 Conclusion ...... 86

CHAPTER FIVE: NUDIBRANCH - SPONGE INTERACTION: FEEDING ECOLOGY AS ASSESSED BY FATTY ACID BIOMARKERS ...... 88 5.1 Introduction...... 88 5.1.1 Hypothesis ...... 91 5.1.2 Aims ...... 91 5.2 Materials and Methods ...... 92 5.2.1 Sample Collection ...... 92 5.2.2 Field Feeding Observations...... 93 5.2.3 Sponge Documentation ...... 94 5.2.3.1 Sponge Morphology ...... 94 5.2.3.2 Nitric Acid Digestion and Spicule Preparation ...... 94

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5.2.3.3 Thick Sectioning ...... 95 5.2.3.4 Sponges Final Identification ...... 95 5.2.4 Fatty Acid Analysis of Two Sponge Species ...... 96 5.2.4.1 Total Lipid Extraction ...... 96 5.2.4.2 Preparation of Fatty Acid Methyl Ester (FAME) ...... 97 5.2.4.3 Statistical Analyses ...... 98 5.4 Results ...... 98 5.4.1 Field Observations ...... 98 5.4.2 Sponge Identification ...... 99 5.4.2.1 Taxonomy ...... 99 5.4.3 Field Feeding Observations...... 104 5.4.4 Fatty Acid Analysis in Sponges ...... 105 5.4.5 Distribution of Fatty Acid Groups in Phyllidia varicosa and its Sponge Prey ...... 110 5.5 Discussion ...... 113 5.5.1 Sponge Species Documentation and their Fatty Acid Contents ...... 114 5.5.2 Distribution of Fatty Acid Groups in Phyllidia varicosa ...... 115 5.5.2.1 Odd- and Branched-chain Fatty Acids ...... 115 5.5.2.2 Comparison of OBFA in Phyllidia varicosa and its Prey Sponge ...... 117 5.2.3 Demospongic Fatty Acids ...... 119 5.6 Conclusion ...... 120

CHAPTER SIX: LABORATORY OBSERVATIONS ON BEHAVIOURAL PATTERNS OF NUDIBRANCH Phyllidia varicosa AND Phyllidiella pustulosa .... 121 6.1 Introduction...... 121 6.1.1 Aims ...... 122 6.2 Materials and Methods ...... 122 6.3 Results ...... 125 6.3.1 Abnormal Behaviours ...... 125 6.3.2 Normal Behaviours ...... 126 6.3.2.1 Mating Behaviour ...... 126 6.3.2.2 Egg Deposition ...... 127 6.4 Discussion ...... 129 6.5 Conclusion ...... 131

CHAPTER SEVEN: GENERAL CONCLUSION AND RECOMMENDATION .. 132 7.1 General Conclusion ...... 132 7.2 Future Study and Recommendation...... 133

REFERENCES ...... 134

APPENDICES ...... 157 APPENDIX A ...... 157 APPENDIX B ...... 161

LIST OF TABLES

Table No. Page No. Table 2.1 Overview and appraisal of three lipid extraction methods 40

Table 2.2 Metabolites isolated from phyllidiid nudibranchs 43

Table 3.1 List of Concentration and Purity of Extracted DNA 52

Table 3.2 Pairwise distance matrix calculated for the nudibranch 54 species

Table 4.1 The average length of the examined species 67

Table 4.2 Weights of the nudibranch samples and total lipid extract 68

Table 4.3 The data arrangement in Excel sheet for the multivariate analysis of fatty acid composition 71

Table 4.4 The data arrangement in Excel sheet for the univariate analysis of fatty acid groups of the nudibranch species 72

Table 4.5 The percentage of fatty acid composition of total FAME from coastal water of Balok and Bidong Island 77

Table 4.6 Total percentages of fatty acid groups of total FAME from Balok and Bidong Island of Phyllidia varicosa and 79 Phyllidiella pustulosa

Table 4.7 Statistical analysis results for the univariate and multiv- 79 ariate tests

Table 4.8 The pairwise analyses on two factors (species and site) of 80 nudibranch fatty acid composition

Table 5.1 Fatty acid composition of marine sponge Xestospongia 107 vansoesti and Aaptos suberitoides % of total fatty acids

Table 6.1 Various parameter ranges for seawater, and ideal ranges 123 for marine husbandry

LIST OF FIGURES

Figure No. Page No. Figure 1.1 The flow chart of the general methodology that used in the 7 study

Figure 1.2 The flow chart of the species identification in the study 8

Figure 1.3 The flow chart of the fatty acids analysis in the study 9

Figure 1.4 The flow chart of the fatty acids analysis for the nudibranch 10 and sponges in the study

Figure 1.5 The flow chart of the sponge identification in the study 11

Figure 2.1 External morphology and colouration of Phyllidia varicosa 18

Figure 2.2 External morphology and colouration of Phyllidiella pustulosa 19

Figure 2.3 the closest appearance of Phyllidiella zeylanica to the 19 Phyllidiella pustulosa

Figure 2.4 Delta nomenclature system 35

Figure 2.5 Omega nomenclature system 36

Figure 3.1 Maps of the coastal waters of Balok-Pahang and Bidong 47 Island-Terengganu-Malaysia

Figure 3.2 1% agarose gel electrophoresis of genomic DNA extraction of 51 nudibranch species

Figure 3.3 Electrophoretic analysis of 0.8% agarose gels containing 16S 52 rDNA gene polymerase chain reaction (PCR) products

Figure 3.4 Neighbour-joining original phylogenetic tree of phyllidiid 56 nudibranchs

Figure 3.5 Maximum likelihood tree of the phyllidiid nudibranchs based 57 on the 16S mtDNA gene Figure 4.1 Maps of the coastal waters of Balok, Pahang and Bidong 64 Island, Terengganu, Malaysia

Figure 4.2 Measurements of the examined species were taken in the 66 laboratory

Figure 4.3 Tubercle size of Phyllidiella pustulosa adults 66

Figure 4.4 Phyllidiella pustulosa juveniles 67

Figure 4.5 Gas chromatogram of the FAME from the total lipids of the 73 Phyllidia varicosa from coastal water of Balok.

Figure 4.6 Gas chromatogram of the FAME from the total lipids of the 74 Phyllidia varicosa from Bidong Island.

Figure 4.7 Gas chromatogram of the FAME from the total lipids of the 75 Phyllidiella pustulosa from coastal water of Balok

Figure 4.8 Gas chromatogram of the FAME from the total lipids of the 76 Phyllidiella pustulosa from Bidong Island

Figure 4.9 Principal coordinate ordination (PCO) of the fatty acid profile 81 from Phyllidia varicosa and Phyllidiella pustulosa

Figure 4.10 nDMS plot of fatty acid distribution between the studied 82 species and sites

Figure 4.11 Total percentages of fatty acids of total FAME from Balok 83 and Bidong Island of Phyllidia varicosa and Phyllidiella pustulosa

Figure 5.1 Maps of the coastal waters of Balok-Pahang and Bidong 93 Island-Terengganu-Malaysia.

Figure 5.2 Viscera and notum of Phyllidia varicosa 97

Figure 5.3 The documentation of the Phyllidia varicosa attaching on its 99 sponge prey in the coastal waters of Balok

Figure 5.4 Underwater photo of sponge species from the coastal water 100 of Balok, species name: Xestospongia vansoesti

Figure 5.5 Spicule morphology (40) of sponge species from the 101 coastal water of Balok, species name: Xestospongia vansoesti.

Figure 5.6 Choanosomal skeleton (40) of sponge species from the 101 coastal water of Balok, species name: Xestospongia vansoesti

Figure 5.7 Underwater photo of sponge species from Bidong Island, 103 species name: Aaptos suberitoides

Figure 5.8 Spicule morphology (40 of sponge species from Bidong 104

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Island, species name: Aaptos suberitoides

Figure 5.9 Phyllidia pustulosa while feeding on sponge 105

Figure 5.10 Gas chromatogram of the FAME from the total lipids of the 108 sponge Xestospongia vansoesti

Figure 5.11 Gas chromatogram of the FAME from the total lipids of the 109 sponge Aaptos suberitoides

Figure 5.12 The mass spectrum of fatty acid 5, 9-25:2 110

Figure 5.13 Composition of odd-numbered carbon chain and branched 111 fatty acids (OBFA)

Figure 5.14 Composition of odd-numbered carbon chain and branched 112 fatty acids (OBFA) in tissues (viscera and notum) of the nudibranch Phyllidia varicosa and its sponge prey

Figure 5.15 Principal coordinate ordination (PCO) of the fatty acid 113 profile from the nudibranch species and Xestospongia vansoesti sponge

Figure 6.1 Nudibranch species: Phyllidia varicosa and Phyllidiella 123 pustulosa

Figure 6.2 Side view of the aquarium 124

Figure 6.3 Glass aquarium used for observing the behaviour of 125 phyllidiid species

Figure 6.4 Unusual behaviour of nudibranch species in an aquarium 126

Figure 6.5 Phyllidia varicosa species lift up their mantle edge 126

Figure 6.6 Mating between two Phyllidia varicosa species 127

Figure 6.7 Egg deposition by Phyllidia varicosa form a counter 128 clockwise spiral Figure 6.8 Egg ribbon of Phyllidiella pustulosa 128

Figure 6.9 Spiral egg formation of Phyllidia varicosa 129

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LIST OF SYMBOLS

bp Base Pair

°C Celsius cm Centimetres h Hour g Gram mg Milligram

µl Microliter m/z Mass-to-charge ratio

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LIST OF ABBREVIATIONS

BLAST Basic Local Alignment Search Tool

CPOA Cyclopropaneoctanoic Acid

EtBr Ethidium bromide

EPA Eicosapentaenoic Acid

DHA Docosahexaenoic Acid

DNA Deoxyribonucleic Acid

IUPAC International Union of Pure and Applied Chemistry

GC-MS Gas Chromatography–Mass Spectrometry

HCl Hydrochloric Acid

FAME Fatty Acids Methyl Ester

KCl Potassium chloride mit DNA Mitochondrial Deoxyribonucleic Acid

MUFA Monounsaturated Fatty Acids

NaCl Sodium chloride

NMDS Non-metric multidimensional scaling

NMID Non-methylene interrupted fatty acids

OBFA Odd- chain Branched Fatty Acids

PUFA Polyunsaturated Fatty Acids

PCO Principal Coordinate Ordination

PCR Polymerase Chain Reaction rDNA Ribosomal Deoxyribonucleic Acid

SFA Saturated Fatty Acids

UV Ultraviolet

VLCFA Very long-chain fatty acids

CHAPTER ONE

GENERAL INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Nudibranch molluscs are one of the most attractive marine organisms and widely distributed in all oceans and seas. There are more than 3,000 known species of different coloured nudibranchs from bright blue and pink to yellow and white with orange dots (Brunckhorst, 1993; Grande et al., 2002; Kubanek et al., 2000; Su et al.,

2009). Nudibranch group has been divided into four main taxa: Aeolidoidea,

Arminoidea, Dendronotoidea and Doridoidea. The family Phyllidiidae is commonly known to be the largest within the dorid nudibranchs. Phyllidiid nudibranchs have been studied by Brunckhorst (1993), who has released the phyllidiid taxonomy. The recently published new findings of the phyllidiid species still follow the same taxonomy (Domínguez et al., 2007; Fahrner and Beck, 2000; Fahrner and Schrodl,

2000a; Fahrner and Schroedl, 2000b; Jung et al., 2014; Valdés and Behrens, 2002;

Valdés and Gosliner, 1999; Yonow, 1996). However, the studies on phyllidiid nudibranch species in tropical ecosystem are still limited and their chemical ecology is particularly appealing and poorly investigated.

Phyllidia varicosa and Phyllidiella pustulosa, which belong to the

Phyllidiidae family, are the most frequently encountered phyllidiid nudibranchs in tropical Indo-Pacific waters (Brunckhorst, 1993). These two species are also quite abundant in Malaysia, such as in coastal waters of Balok, Pahang and Bidong Island,

Terengganu. In general, Phyllidiidae family individuals are similar in the appearance and this complicates the visual identification. The existing studied species can be

1 distinguished from their conspecifics through their external morphology, such as the pattern of the mantle and rhinophores.

Most of the identification approaches for the nudibranch molluscs use morphological and anatomical characteristics because of some barriers to DNA isolation, such as the type of tissue (e.g., mantle, adductor muscle, foot muscle or gills) and its physiology. There are some obstacles to the DNA extraction process that might contribute to the lack of genetic research in nudibranchs compared with other marine species (Pereira et al., 2011; Winnepenninckx et al., 1994). The similar appearance of phyllidiid nudibranch species might complicate visual identification.

Therefore, the genetic approach to species identification was very important to confirm that the studied species were identical. A part of mitochondrial 16S rDNA is easily amplified using universal primers and has been applied to species identification and phylogenetic studies in various groups and at different levels (De Masi et al.,

2015; Han and Mcpheron, 1997; Pola et al., 2007; Valdés, 2003; Wilson and Lee,

2005; Wollscheid et al., 2001). Therefore, a correct identification tool for the studied nudibranch species may help to facilitate subsequent analyses of nudibranch species.

Fatty acids with great diversity, varying biochemical structures, and unique origins among marine organisms have enhanced a number of areas of research interest, ranging from estimating animal nutrition and metabolism to investigating trophic interactions (Budge, 2006). Thus, fatty acids have been used as a valuable indicator to differentiate molluscan species and environmental conditions (Galap et al., 1999). Therefore, fatty acid composition was used to indicate the dietary status of selected species of P. varicosa and P. pustulosa and to compare the fatty acid contents among members of the same species from two different areas.

Phyllidiid nudibranchs might have specific food preferences and they are considered one of the most important organisms that feed on sponges. The sponge- feeding nudibranchs are able to adapt to the venomous sponges chemicals and are also able to sequester these harmful chemicals from their diet and utilize them as defensive chemicals (Kubanek et al., 2000; Lyakhova et al., 2010). In addition, Zhukova, (2014) found that a high level of very long-chain fatty acids, called demospongic acids, in reference to their food habitats. These fatty acids were found in different concentrations that are specific to the sponge species. Therefore, the investigation on fatty acid composition in phyllidiid nudibranchs in related to its sponge prey could contribute to additional evidence of the trophic relationship between the prey and predator.

The behaviour of marine organisms is poorly investigated due to the difficulties accessing their natural habitat. In nudibranchs, mating behaviour and reproduction processes have been studied under laboratory conditions, for example, in the simultaneous hermaphrodite nudibranch molluscs Aeolidiella glauca, due to the difference between A. glauca and other nudibranchs (Karlsson & Haase, 2002).

However, in phyllidiid nudibranchs, less information is available about the behaviour of this family. Here, field feeding observations were conducted during frequent sampling and the mating behaviour, egg deposition and other abnormal behaviours were observed under laboratory conditions.

1.2 PROBLEM STATEMENT

The diversity in nudibranch species in a particular zone may serve as a good sign of ecosystem health. Researchers have reported that nudibranch species seem to have close associations with other aquatic invertebrates as signs of marine ecosystem imbalance (Edgar, 2000; Goddard et al., 2011a; O'Hara, 1995). To date, there have been few documented studies about nudibranch biology in Malaysia despite its important impact on the marine ecosystem.

In this study, size variation in nudibranchs species from two different areas was observed. This variation triggers questions surrounding the phylogenetic position, primary metabolites, and feeding ecology of these species. In general, the chemical ecology of phyllidiid nudibranchs is particularly appealing and their fatty acid composition is poorly investigated. Therefore, analysis of the fatty acid composition of marine invertebrates could be valuable in determining their food source. As far as we know, there are few deliberate ecological studies available on the phyllidiid nudibranch group and their interaction with prey. In addition, documentation of the feeding ecology and the use of fatty acids to reveal the phyllidiid diet status are scarce.

Investigations on fatty acids as biomarkers in the studied species in related to its sponge prey are also poorly explored. In addition, the behaviour of the phyllidiidae family is poorly investigated due to the difficulty of accessibility to the natural habitat.

Therefore, these aspects of the research would be vital to better solve this concern.

1.3 RESEARCH HYPOTHESIS

 There are ecological and phylogenetic factors which lead to size variation in

nudibranchs species.

 There are qualitative and quantitative variations in fatty acid composition

depending on diet specificity.

1.4 OBJECTIVES OF THE STUDY

1- To determine the molecular phylogeny of the selected nudibranch species and their closest genetic relatives collected from coastal waters of Balok (Pahang) and Bidong

Island (Terengganu), Malaysia using the mitochondrial large ribosomal DNA gene

(16S rDNA) as a target reference sequence.

2- To investigate and compare the composition of fatty acids in Phyllidia varicosa and

Phyllidiella pustulosa collected from two different locations.

3- To understand the feeding ecology and food preferences of P. varicosa at its nesting grounds.

4- To investigate the behaviour of the selected nudibranch species, such as mating behaviour and egg deposition, and the abnormal behaviour patterns that can be found in the laboratory.

1.5 SIGNIFICANCE OF THE STUDY

Nudibranchs are ecologically important as an indicator of a healthy ecosystem. A decrease in their species diversity in a particular area can be an early warning signal of diminished environmental quality (Goddard et al., 2011b). However, nudibranch organisms have been overlooked in deliberate ecological studies. Therefore, knowledge about nudibranchs is essential in order to understand their biology and the surrounding environment.

The data presented here will contribute to our understanding of the combination between molecular and morphological data for species identification.

Furthermore, using 16S rDNA as a genetic marker simplifies the identification of species accurately up to the species level. Analysis of the fatty acids and the effect of diet specificity on nudibranch fatty acid content provide insight into the nudibranch- sponge interaction indicated by fatty acid biomarkers. Findings from this research will provide reliable scientific data about the role of demospongic acids in the trophic relation between nudibranchs and sponges.

1.6 GENERAL RESEARCH METHODOLOGY

This part provides information on the research methods of this thesis that used to achieve the aims of the study. Samplings were performed frequently and all samplings were done by mean of SCUBA at the selected study areas of coastal water of Balok,

Pahang and Bidong Island, Terengganu, Malaysia. In general, nudibranch samples were transferred alive in a plastic box equipped with live rock and aeration pumps and then immediately transported to International Islamic University Malaysia (IIUM).

The experiments were divided into three parts as shown in figure 1.1.

Figure 1.1 The flow chart of the general methodology that used in the study.

The first part of the study included the identification of nudibranch species from coastal waters of Balok (Pahang) and Bidong Island (Terengganu), Malaysia. In

7 this part, the genomic DNA was extracted by using QIAGEN kit and the targeted gene was amplified by using universal primers and the PCR products were sent for sequencing for the phylogenetic analysis (Figure 1.2). To study the phylogenetic relationships within the Phyllidiidae, mitochondrial 16S rDNA sequences of selected species was aligned against corresponding sequences of phyllidiid nudibranchs reported previously and retrieved from the NCBI nucleotide database.

Figure 1.2 The flow chart of the species identification in the study.

The second part of the study focused on the fatty acids analysis for the nudibranch and sponges, and this included total lipids extraction, esterification, and analysis by using Gas chromatography–mass spectrometry (GC-MS) as shown in figure 1.3. The experiment was designed to reveal the fatty acid composition in the studied nudibranch and to compare the composition in two different locations.