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Biochemical Properties of Anemone Fish Mucous to Understand Its Adaptation to Sea Anemone

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Biochemical Properties of Anemone Fish Mucous to Understand Its Adaptation to Sea Anemone BIOCHEMICAL PROPERTIES OF ANEMONE FISH MUCOUS TO UNDERSTAND ITS ADAPTATION TO SEA ANEMONE BY NAJATUL SU AD BINTI ABDULLAH A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biosciences) Kulliyyah of Science International Islamic University Malaysia JANUARY 2016 ABSTRACT The study of anemone fish mucous to understand anemone fish adaptation to sea anemone was aimed to identify the biochemical composition in the fish mucous that enabled the protection of anemone fish against the stinging tentacles of sea anemones. Upon sampling, one species of sea anemone, Heteractis magnifica was identified with its resident anemone fish, the false clownfish, Amphiprion ocellaris, along with 2 other non-symbiont coral reef fish species, the scissor-tailed sergeant, Abudefduf sexfasciatus (Family Pomacentridae) and the moon wrasse, Thalassoma lunare (Family Labridae) to provide comparison and knowledge insight into anemone fish ability to live unharmed along the tentacles of sea anemone, H. magnifica. The scissor-tailed sergeant and the moon wrasse are among many other coral reef fishes that are observed to be stung upon contact with the sea anemone tentacles that elicit nematocyst discharge. Mucous of false clownfish, A. ocellaris was extracted and investigated for its glycoprotein by studying the protein and sialic acid content and compared against the mucous content of sea anemone, H. magnifica, and other fish species, the scissor-tailed sergeant, A. sexfasciatus and the moon wrasse, T. lunare. Protein identification was done by SDS-PAGE and MALDI-TOF analysis while thiobarbituric acid assay followed by HPLC detection were performed to assess sialic acid content in the mucous. Results indicated that the false clownfish had keratin and actin glycoproteins mucous with 1.636 mg/ml sialic acid content while for non-symbiont fish, the scissor-tailed sergeant presented collagen glycoprotein with 50.433 mg/ml concentration of sialic acid and moon wrasse had high molecular weight proteins with 71.893 mg/ml sialic acid content. The higher concentration of sialic acid was involved in causing the tentacles of sea anemone to discharge toxin. Hence, it is concluded that the false clownfish could afford protection from the stinging toxins of sea anemone by having glycoproteins with very low content of sialic acid which was not adequate to trigger sea anemone response for toxin discharge. ii خﻻصة البحث ABSTRACT IN ARABIC إن دراسة خماط مسكة املهرج ﻹدراك قابلية التكيف مسكة املهرج لشقائق البحر هتدف إىل حتديد تركيبة كيميائية حيوية ملخاط مسكة املهرج الذي حيميها من لسعة جمس شقائق البحر. أثناء اﻻختبار، تُستخدم إحدى شقائق البحر، هيترياكتيس ماغنيفيكا Heteractis magnifica ,ومسكة املهرج املِقيمة هبا، أوسيﻻريس أمفيربيون Amphiprion ocellaris، مع نوعني من السمكة املرجانية غري متعايشتني، الرقيب سسيسورتيل scissortail sergeant، أبو دفدوف سيكسفاسسياتوس Abudefduf sexfasciatus )فصيلة بوماسينرتيداي Pomacentridae( وُمن الراس moon wrasse، تﻻسوما لوانري Thalassoma lunare)فصيلة اللربوسية Labridae( مع كوهنا دراسة مقارنة وإملام اتم بقدرة مسكة املهرج على العيش ساملات بني جمس شقائق البحر هيترياكتيس ماغنيفيكا .H magnifica. بطبيعة احلال، يﻻحظ أن الرقيب سسيسورتيل scissortail sergeant وُمن الراس moon wrasse من بني اﻷمساك املرجانية اﻷكثر ملدوغة عند اﻻحتكاك مبجس شقائق البحر اليت تنتزع كيسة ﻻسعة. يتم البحث ابستخراج خماط أوسيﻻريس أمفيربيون A. ocellaris وفحص بروتينها السكري بطريقة دراسة مضمون الربوتني ومحض السياليك ومقارنته مبضمون خماط شقائق البحر، هيترياكتيس ماغنيفيكا H. magnifica وكذلك مضمون السمكتني املرجانيتني الرقيب سسيسورتيل scissortail sergeant، أبو دفدوف سيكسفاسسياتوس A. sexfasciatus وُمن الراس moon wrasse، تﻻسوما لوانري T. lunare. وأجرى فحص برادفورد للربوتني Bradford assayوالفصل الكهرابئي هلﻻم كربيتات دوديكل الصوديوم متعدداﻷكريﻻمايد SDS-PAGE يف حتقيق الربوتني، ومن مث أجرى اختبار محض ثيوابربيتورات وعملية كروماتوغرافيا سائلة عالية اﻷداء HPLCلتقييم مضمون محض السياليك خلﻻصات املخاط. وتشري النتيجة إىل أن مسكة املهرج هلا وزن جزيئي منخفض وقلة مضمون محض السياليك. وأما السمكتان املرجانيتان غري متعايشتان تتميزان بوزن جزيئي مرتفع وكثرة مضمون محض السياليك. فمن هنا يتضح أن محض السياليك يسبب نزع جمس شقائق البحر ذيفاهنا. ومن هنا، نستنتج أبن مسكة املهرج قادرة على محاية نفسها ضد لسعة جمس شقائق البحر ِ املسمومة إثْ َر تضمنها بروتني السكري دون محض السياليك. iii 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). ………………………………….. Shahbudin Saad Main Supervisor ………………………………….. Deny Susanti 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). ………………………………….. Tengku Haziyamin Tengku Abd. Hamid Examiner This thesis was submitted to the Department of Biotechnology and is accepted as a partial fulfilment of the requirements for the degree of Master of Science (Biosciences). ………………………………….. Suhaila Mohd Omar Head, Department of Biotechnology This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of the requirements for the degree of Master of Science (Biosciences). ...................................................... Kamaruzzaman Yunus Dean, Kulliyyah of Science iv 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. Najatul Su Ad Binti Abdullah Signature ........................................................... Date ......................................... v INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH BIOCHEMICAL PROPERTIES OF ANEMONE FISH MUCOUS TO UNDERSTAND ITS ADAPTATION TO SEA ANEMONE I declare that the copyright holder of this thesis is International Islamic University Malaysia. Copyright © 2015 by International Islamic University Malaysia. All rights reserved. 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 retrieval 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 Najatul Su Ad Binti Abdullah. ……………………………. ……………….. Signature Date vi ACKNOWLEDGEMENTS All Praises to Allah, Most Gracious and Most Merciful. This thesis was possible with the supervision of Assoc. Prof. Dr. Shahbudin Sa’ad and Assoc. Prof. Dr. Deny Susanti, to whom both I owe my lifetime of gratitude for their guidance, patience and encouragements throughout the research. I would like to acknowledge the scholarship of IIUM for my appointment as an Academic Trainee. Thank you to all staff of Kulliyyah of Science and friends that helped me greatly in my research works and study period here including Br. Azizul Aziz, Br. Muhammad Faiz, Miss Nasihin Seth, Br. Amir, Mrs. Fikriah Faudzi, Br. Fuad Miskon, Sr. Juliana, Br. Zaini, Br. Fikri Akmal, Br. Hamizan, Br. Husaini, Miss Nor Shuhadah Warzukni, Br. Muzzamil, Miss Aminah Kaida, Miss Hafizah Zakaria, Miss Amira Farzana, Br. Hendy Putra, Br. Jazli Aziz, Br. Fuad Anuar, Miss Amalina Soudin and many more. I would also like to express my heartfelt apologies and appreciation to my family; my mum, Mrs Faridah Hamdan, my dad, Mr Abdullah Sadli, my in-laws, Mr Mohd Sidek and Mrs Zawiah, my darling husband, Mr. Ahmad Mustaqim, and my baby boy who grew with this thesis, Ahmad Amsyar, for giving me strength when I felt weak and believed that I could accomplish this. Last but not least, my daughter, Mawar Hani, whom I delivered with this thesis. Thank you. vii TABLE OF CONTENTS Abstract .................................................................................................................... ii Abstract in Arabic .................................................................................................... iii Approval Page .......................................................................................................... iv Declaration ............................................................................................................... v Copyright Page ......................................................................................................... vi Acknowledgements .................................................................................................. vii List of Tables ........................................................................................................... x List of Figures .......................................................................................................... xi CHAPTER ONE : INTRODUCTION ................................................................
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