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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
‫خالصة البحث‬
‫‪ABSTRACT IN ARABIC‬‬
‫إن دراسة خماط مسكة املهرج إلدراك قابلية التكيف مسكة املهرج لشقائق البحر هتدف إىل حتديد تركيبة‬
‫كيميائية حيوية ملخاط مسكة املهرج الذي حيميها من لسعة جمس شقائق البحر‪ .‬أثناء االختبار‪ ،‬تُستخدم‬
‫إحدى شقائق البحر‪ ،‬هيترياكتيس ماغنيفيكا ‪, Heteractis magnifica‬ومسكة املهرج ِ‬
‫املقيمة هبا‪،‬‬
‫أوسيالريس أمفيربيون ‪ ،Amphiprion ocellaris‬مع نوعني من السمكة املرجانية غري متعايشتني‪،‬‬
‫الرقيب سسيسورتيل ‪ ،scissortail sergeant‬أبو دفدوف سيكسفاسسياتوس ‪Abudefduf‬‬
‫ومن الراس ‪،moon wrasse‬‬
‫‪( sexfasciatus‬فصيلة بوماسينرتيداي ‪ُ )Pomacentridae‬‬
‫تالسوما لوانري ‪(Thalassoma lunare‬فصيلة اللربوسية ‪ )Labridae‬مع كوهنا دراسة مقارنة‬
‫وإملام اتم بقدرة مسكة املهرج على العيش ساملات بني جمس شقائق البحر هيترياكتيس ماغنيفيكا ‪H.‬‬
‫ومن الراس‬
‫‪ .magnifica‬بطبيعة احلال‪ ،‬يالحظ أن الرقيب سسيسورتيل ‪ُ scissortail sergeant‬‬
‫‪ moon wrasse‬من بني األمساك املرجانية األكثر ملدوغة عند االحتكاك مبجس شقائق البحر اليت‬
‫تنتزع كيسة السعة‪ .‬يتم البحث ابستخراج خماط أوسيالريس أمفيربيون ‪ A. ocellaris‬وفحص بروتينها‬
‫السكري بطريقة دراسة مضمون الربوتني ومحض السياليك ومقارنته مبضمون خماط شقائق البحر‪،‬‬
‫هيترياكتيس ماغنيفيكا ‪ H. magnifica‬وكذلك مضمون السمكتني املرجانيتني الرقيب سسيسورتيل‬
‫ومن الراس‬
‫‪ ،scissortail sergeant‬أبو دفدوف سيكسفاسسياتوس ‪ُ A. sexfasciatus‬‬
‫‪ ،moon wrasse‬تالسوما لوانري ‪ .T. lunare‬وأجرى فحص برادفورد للربوتني ‪Bradford‬‬
‫‪assay‬والفصل الكهرابئي هلالم كربيتات دوديكل الصوديوم متعدداألكريالمايد ‪ SDS-PAGE‬يف‬
‫حتقيق الربوتني‪ ،‬ومن مث أجرى اختبار محض ثيوابربيتورات وعملية كروماتوغرافيا سائلة عالية األداء‬
‫‪HPLC‬لتقييم مضمون محض السياليك خلالصات 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 إِثْ َ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 ...........................................................
v
Date .........................................
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
vi
………………..
Date
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 ................................................................. 1
1.1 Background of the Study ........................................................................ 1
1.1.1 Benefit of Study ............................................................................ 4
1.2 Research Question .................................................................................. 4
1.3 Research Hypothesis ............................................................................... 4
1.4 Research Objective ................................................................................. 5
1.4.1 General Objective ......................................................................... 5
1.4.2 Specific Objectives ....................................................................... 5
CHAPTER TWO : LITERATURE REVIEW .................................................... 6
2.1 Anemone Fish Background Study .......................................................... 6
2.1.1 False Clownfish............................................................................. 9
2.2 Symbiosis with Sea Anemone ................................................................ 11
2.3 Sea Anemones ........................................................................................ 13
2.3.1 Magnificent Sea Anemone ............................................................ 13
2.4 Non-Symbiont Fishes ............................................................................. 14
2.4.1 Scissor-Tailed Sergeant................................................................. 15
2.4.2 Moon Wrasse ................................................................................ 16
2.5 Mucous Biochemical Composition ........................................................ 18
2.5.1 Fish Mucous Glycoprotein ............................................................ 19
2.5.1.1 Protein Determination....................................................... 20
2.5.1.2 Sialic Acids ....................................................................... 20
CHAPTER THREE : METHODOLOGY........................................................... 23
3.1 Experimental Materials ........................................................................... 23
3.1.1 Sample Collection ......................................................................... 23
3.1.1.1 Location of specimen collection ....................................... 23
3.1.1.2 Specimen Species Identification ....................................... 26
3.1.1.3 Sample Collection............................................................. 26
3.1.2 Chemicals and Reagents ............................................................... 26
3.1.3 Equipment and Instruments .......................................................... 27
3.1.4 Consumable Items ......................................................................... 27
3.1.5 Glassware ...................................................................................... 27
3.1.6 Sterilization ................................................................................... 27
3.2 Experimental Methods ............................................................................ 28
3.2.1 Sea Anemone Mucous Extraction ................................................. 28
viii
3.2.2 Fish Mucous Extraction ................................................................ 28
3.3 Analytical Method .................................................................................. 29
3.3.1 Bradford Assay ............................................................................. 29
3.3.2 Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis
(SDS-PAGE) ................................................................................ 30
3.3.2.1 Visualisation of Bands ...................................................... 31
3.3.3 Matrix-Assisted Laser Desorption Ionization Time-of-Flight
(MALDI-TOF) mass spectrometry analysis ................................. 31
3.3.4 Sialic Acid Determination ............................................................. 32
3.3.4.1 Statistical Analysis ........................................................... 33
3.4 Ichthyotoxicity assay .............................................................................. 33
CHAPTER FOUR35 : RESULTS AND FINDINGS .......................................... 35
4.1 Specimen Species Identification ............................................................. 35
4.1.1 Sea Anemone Identification and Taxonomy ................................. 35
4.1.2 Fish Species Identification and Taxonomy ................................... 36
4.2 Estimating The Protein Purification By Bradford Assay ....................... 39
4.3 Estimating Protein Molecular Weight Through SDS-PAGE ................. 40
4.4 MALDI-TOF Mass Spectrometry Search Analysis ............................... 43
4.5 Sialic Acid Determination ...................................................................... 46
4.5.1 Sea Anemone ................................................................................ 48
4.5.2 False Clownfish............................................................................. 49
4.5.3 Scissor-tailed Sergeant .................................................................. 50
4.5.4 Moon Wrasse ................................................................................ 51
4.6 Ichthyotoxicity assay .............................................................................. 53
CHAPTER FIVE : DISCUSSIONS ..................................................................... 56
5.1 Sample Selection and Species Identification .......................................... 56
5.1.1 Sample Selection ........................................................................... 56
5.1.2 Species Taxonomic Identification ................................................. 56
5.1.2.1 Fish Species Identification................................................ 57
5.1.2.2 Sea Anemone Species Identification ................................ 58
5.2 Protein Detection and Determination In Mucous Extracts ..................... 59
5.2.1 Sea Anemone Mucous Extract ...................................................... 61
5.2.2 False Clownfish Mucous Extract .................................................. 62
5.2.3 Scissor-tailed Sergeant Mucous Extract ....................................... 63
5.2.4 Moon Wrasse Mucous Extract ...................................................... 64
5.3 Sialic Acid Determination of Mucous Extracts ...................................... 65
5.4 False Clownfish Adaptation to Sea Anemone ........................................ 67
CHAPTER SIX : CONCLUSION AND FUTURE WORKS ............................ 69
6.1 Conclusion .............................................................................................. 69
6.2 Future Works .......................................................................................... 70
BIBLIOGRAPHY ................................................................................................. 71
APPENDIX A : DATA FOR PROTEIN ANALYSIS ...................................... 77
APPENDIX B : MASS SPECTRA OF PEPTIDE SEQUENCES ................... 81
ix
LIST OF TABLES
4.1
Taxonomy and External Morphology of Sea Anemone
36
4.2
Taxonomy and External Morphology of Fishes
36
4.3
Protein Purification from Sample Extraction.
39
4.4
Molecular Weights of Visualised Bands from SDS-PAGE.
43
4.5
Search Hits of Peptide Fragments from Sea Anemone
43
Mucous Sample.
4.6
Search Hits of Peptide Fragments from False Clownfish
44
PAGE Gel Band 1.
4.7
Search Hits of Peptide Fragments from False Clownfish
45
PAGE Gel Band 2.
4.8
Search Hits of Peptide Fragments from Scissor-Tailed
46
Sergeant PAGE Gel Band 1.
4.9
Sialic Acid Standard Concentration and Peak Area for
46
Plotting Standard Curve.
4.10
Mean of Sialic Acid Content Concentration.
52
4.11
Fish Response to Sea Anemone Extract.
53
4.12
Response of Sea Anemone to Fish Extract and NANA.
55
x
LIST OF FIGURES
2.1
A Photograph of a Common False Clownfish in Malaysian
Waters Contributed by Muhammad Faiz, Dept of Marine
Science, IIUM.
10
2.2
Geographical Distribution of False Clownfish Population.
Retrieved September 15, 2013, from Fishbase Website:
Http://Www.Fishbase.Org/Summary/6509.
10
2.3
A Photograph of a Common H. Magnifica in Malaysian
Waters Contributed by Muhammad Faiz, Dept Of Marine
Science, IIUM.
14
2.4
A Photograph of a Common Scissor-Tailed Sergeant,
A. Sexfasciatus, in Malaysian Waters Contributed by
Muhammad Faiz, Dept Of Marine Science, IIUM.
15
2.5
A Photograph of a Common Moon Wrasse, T. Lunare, in
Malaysian Waters.
17
3.1
Map of Sample Site 1, Balok.
24
3.2
Map of Sample Site 2, Tioman.
25
4.1
Specimen Photographed from Above Prior to Collection.
Photograph was Contributed by Muhammad Faiz, Dept of
Marine Science, IIUM.
35
4.2
Specimen Photographed on Board Prior to Mucous Sample
Collection and Release Back to the Sea. Photograph was
Contributed by Muhammad Faiz, Dept of Marine Science,
IIUM.
37
4.3
Specimen Photographed on Board Prior to Mucous Sample
Collection and Release Back to the Sea. Photograph was
Contributed by Fikri Akmal, Dept of Marine Science, IIUM.
38
4.4
Specimen Photographed on Board Prior to Mucous Sample
Collection and Release Back to the Sea. Photograph was
Contributed by Fikri Akmal, Dept of Marine Science, IIUM.
38
4.5
Electrophoretic Patterns of Proteins of Sea Anemone
Sample on SDS-PAGE Stained with Coomassie Blue G250.
40
xi
41
4.6
Electrophoretic Patterns of Proteins of False Clown
Anemone Fish on SDS-PAGE Stained with Coomassie Blue
4.7
Electrophoretic Patterns of Proteins of Scissor-Tailed
Sergeant Sample on SDS-PAGE Stained with Coomassie
Blue G-250.
4.8
Electrophoretic Patterns of Proteins of Moon Wrasse
Sample on SDS-PAGE Stained with Coomassie Blue G250.
4.9
Standard Curve for the Quantification of Sialic Acid in
Sample Extracts.
4.10
HPLC Chromatogram of Sialic Acid Detected in Sea
Anemone Mucous Extract Sample.
4.11
HPLC Chromatogram of Sialic Acid Detected in Anemone
Fish Mucous Extract Sample.
41
42
47
48
49
4.12
50
HPLC Chromatogram of Sialic Acid Detected in Scissortail
Sergeant Mucous Extract Sample.
4.13
51
HPLC Chromatogram of Sialic Acid Detected in Moon
Wrasse Mucous Extract Sample.
4.14
52
Graph of One-Way ANOVA for Sialic Acid Content
Comparison
4.15
54
Graph of Repeated Measures One-Way ANOVA for Fish
Response to Sea Anemone Extract Comparison.
4.16
55
Graph of Two-Way ANOVA for Sea Anemone Response
Comparison.
5.1
59
Comparison of Protein Concentration from Sample Crude
and Extracts.
xii
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Anemone fishes are brightly coloured coral reef fishes classified under the damselfish
group (Family Pomacentridae) and named such for its association with sea anemone.
This association have been studied for various aspects such as occurrences and natural
habitat since it was first reported observed in nature by Dr. Cuthbert Collingwood in
1868 (Randall & Fautin, 2002). The fish habitually swims toward the tentacles of sea
anemone and appears unperturbed though other organisms or fishes are stung upon
adherence of the sea anemone tentacles. This lies the uniqueness of the symbiosis
between the anemone fish and sea anemone.
Initially only anemone fish was thought to benefit from the symbiotic
relationship with sea anemone host by protection from predation and as a nesting ground
but research into the field has shown that the sea anemone host also benefits from the
presence of the anemone fish.
It has been documented that anemone fish does
oxygenate its host (Szczebak, Henry, Al-Horani, & Chadwick, 2013) while provides
nutrient and water circulation to encourage growth (Holbrook & Schmitt, 2004).
Recent research on anemone fish symbiosis focus on its physiological make up,
genetic divergence of the anemone fish species, geographical distribution, its host
imprinting and biochemistry relationship between the fish and its host. This indicated
that many still query how the symbiosis is enabled. Many researches revolved around
the question whether the protection of anemone fish from the toxins of sea anemone is
naturally innate or could be acquired through acclimation process, though a generally
1
accepted opinion held that the fish could have afford protection from the stinging
nematocysts by a biochemical constitution in their mucous layer.
Previously Lubbock (1980) showed that one species of anemone fish, the Clark
clownfish, Amphiprion clarkii (Bennett, 1830) mucous layer appeared to be three to
four times thicker than that of related fishes that do not inhabit anemones and consists
largely of glycoprotein. Lubbock (1981) further tested biological compounds to a sea
anemone species that host A. clarkii, the Stichodactyla haddoni. He reported that
protein and glycoprotein elicited strong response while polysaccharide and lipid gave
weaker response. However, no other research on other species of anemone fishes was
done to enable generalization of anemone fishes mucous composition or any other
compound responsible for the protection.
The uniqueness of the symbiotic relationship is further heighten by the
knowledge that only 10 species of sea anemone is reported to support anemone fish
presence while there are more than 800 species of sea anemone discovered worldwide
(Roopin & Chadwick, 2009). Sea anemones are classified under order Actiniaria, class
Anthozoa, phylum Cnidaria, which characteristically possess nematocyst that release
toxins when triggered. Toxins from various sea anemone species known to host
anemone fish and non-host have been elucidated. Significance of these discoveries lead
to enhancement of medicinal therapies
Additionally, the geographical distribution of anemone fishes and sea anemones
hosting symbiont fish is abundant in coastal waters surrounding Malaysia. Even though
sea anemones are found worldwide only the sea anemones found in tropical waters host
anemone fish. Malaysia’s tropical seas is situated in the convergence of Andaman Sea,
South China Sea, and Sulawesi Sea, which are the boundaries between the Indian Ocean
and the Pacific Ocean. That puts Malaysian waters in the Coral Triangle area which
2
although includes countries such as Philippines and Indonesia, it is an area with the
world’s highest marine biodiversity.
Its marine area which spans 351,000 km2 (Spalding, Ravilious, and Green,
2001), with 3,600 km2 coral reef area, more than 350 species of coral identified and to
an approximate of 900 coral reef fish species, which boost prominent fish groups as
damselfishes, anemone fishes, labrids, butterflyfishes, groupers, moray eels and even
the elusive triggerfish. Malaysia nurtures the richest biodiversity of coral reefs, seagrass
beds, mangrove forests and other important habitats in the world making it beneficial
as the research area and may further enhance Malaysia’s recognition in natural marine
resources.
Due to the lack of literature on the isolation of chemical compound from
anemone fish that is directly involved in its host symbiosis, this project aims to find the
compound that may contribute towards further comprehension of co-existence of
anemone fishes with sea anemones. The coasts of Malaysia have high anemone fish
species richness and also a globally valuable area of fish biodiversity, thus provide a
good research site for this project.
In this study, one species of sea anemone hosting anemone fish, one species of
its symbiont anemone fish, and two non-symbiont fish species were identified during
sampling collection. Mucous extracts obtained from the sea anemone and three species
of fish were investigated by the composition of glycoprotein through Bradford assay,
SDS-PAGE and MALDI-TOF techniques for protein identification and thiobarbituric
acid assay followed by HPLC detection for sialic acid determination. The two nonsymbiont fish species identified in the Family Pomacentridae and Family Labridae were
used as comparison throughout the test. Ichthyotoxicity test by which the survival of
3
the fish against host anemone toxin was observed and also sea anemone response to
sialic acid was carried out.
1.1.1 Benefit of Study
Anemone fishes have been the interest of many, researchers, divers and nature lovers
alike. Increasing the knowledge gap in the anemone fishes research is one of the many
benefits of this study. Details on anemone fish mucous provide basic information for
further characterisation of the fish. With the current advancement in scientific research,
previous knowledge on anemone fishes was assayed with new techniques. This study
was imperative as the species originated in Malaysia coastal waters were different than
those found in literature
1.2 RESEARCH QUESTION
How does the protein from the anemone fish mucous differ from other coral reef fish
that enables protection from sea anemone stinging?
1.3 RESEARCH HYPOTHESIS
The mucous coat of anemone fish contains a protein with a different composition
specifically the sialic acid, a biochemical compound that protects the fish against the
toxin sting of sea anemones.
4
1.4 RESEARCH OBJECTIVE
1.4.1 General Objective
To determine the protein and sialic acid component in the anemone fish mucous coat
glycoprotein and to understand how the anemone fish in symbiosis with sea anemone
is protected against stinging.
1.4.2 Specific Objectives
1. To identify selected sea anemone species, anemone fish species and non-symbiont
fish species.
2. To detect and determine the protein content from the mucous of Amphiprion sp.,
other non-symbiont fish species and sea anemone
3. To determine and compare the sialic acid content from the mucous of Amphiprion
sp., other non-symbiont fish species and sea anemone.
5
CHAPTER TWO
LITERATURE REVIEW
2.1 ANEMONE FISH BACKGROUND STUDY
Anemone fishes are among the many brightly coloured fishes of the tropical coral reef
habitat that fascinates the marine environment. Anemone fishes are classified under
phylum Chordata for animals with tissue, coelom, dorsal, tubular nerve cord, and post
anal tail, class Actinopterygii ray-finned spiny fish, order Perciformes for oval shaped
body laterally compressed, and family Pomacentridae for damselfishes.
Anemone fishes are easily recognisable by the striking colour patterns. For
example, the three anemone fishes, Amphiprion ocellaris, A. percula and A. clarkii,
have the characteristically famous orange white vertical bands. Differences between
the three species can be identified by other features such as the size of the bands, with
black markings, and number of spines in their dorsal and anal fin. Knowledge on their
geographical distribution also assists in species identification as existence of
A. ocellaris with its nearest species sibling, A. percula do not overlap (Timm, 2008).
Anemone fishes are also recognisable by their distinctive behaviour swimming
near sea anemones and retreating into the sea anemone tentacles upon caution of danger
or a predator attempts to attack. This lays the uniqueness of the anemone fish as it is
unharmed or appears undisturbed by the tentacles of the sea anemones because the
tentacles are known to possess stinging capsules, nematocyst, that discharges toxin to
prey or predator that approaches near the sea anemone.
Many researches have marvelled the anemone fish ability to live among the sea
anemone. Some have isolated and characterized the toxins of various sea anemones,
host and non-host to fish, as part to understand this symbiosis. Sea anemones are
6
generally found in marine environments around the world but due to the geographical
distribution of anemone fishes in tropical coastal water regions, only 10 species of sea
anemones are known to host fish (Fautin & Allen, 1992).
Other researchers studied on the anemone fishes, its genetic diversity (Timm,
2008), its chemical biology (Mebs, 2009), its behaviour in response to host (Elliott &
Mariscal, 1997) among many strategies to document and explain the mechanism by
which the anemone fish adapts to sea anemones. The mostly applicable opinion is that
these fish have a biochemical composition in their mucous layer that provides protection
from the toxins or stinging tentacles.
However, the conflicting opinions are on the source of the protective mucous
coating whether it is inborn or assimilated. To date, two hypotheses are generally
believed by which the symbiosis may occur. Either the fish essentially produces its own
mucous coat with biochemical compounds to protect itself from the stinging toxins of
the sea anemone tentacles or otherwise, the fish alters the biochemical compounds in
its mucous coat after a behavioural process by which the fish acclimatised to the host
sea anemones.
Lubbock in 1980 tried to research on the base of the first hypothesis that Clark’s
anemone fish, A. clarkii mucous contained the answer to the mechanism that enable the
symbiont-host relationship. He studied the thickness of the mucous, estimated protein
by electrophoresis and used Stichodactyla haddoni for radiolabelling of nutrient
transfer. He concluded that though the fish mucous appears thicker than other nonsymbiotic fish and traces of the sea anemone mucous is found in the mucous coat of the
fish, A. clarkii is protected from stinging by its external mucous layer that is not
fundamentally dependent upon the host.
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Lubbock (1981) further quoted Schlicter (1976) who used radiochemicals and
isoelectric focusing with “inhibitory substances are produced by anemone and surface
of the fish do not have protecting substance”. This raises the question whether the fish
contained substances to protect itself against the toxin or its mucous mimics the sea
anemones mucous to not induce nematocyst discharge. Different groups of compound
were tested and Lubbock (1981) reported that protein and glycoprotein elicited strong
response from S. haddoni while polysaccharide and lipid stimulated the nematocyst less.
However, the nature of the substances in the fish mucous that cause or inhibit response
remains unclear.
On the second hypothesis, Brooks and Mariscal (1984) attempted to understand
the acclimation process by using rubber band fashioned as tentacles attached to rock as
surrogate anemone. They cited works of Martin in 1968 who reported that fish pick up
species specific antigen of anemone and Foster in 1975 who reported that fish produce
protein after acclimation which was not present in non-acclimated fish or anemone
toxin. They concluded their findings by which the anemone fish alters the mucous coat
during acclimation but the presence of surrogate anemone decreases acclimation.
Elliot and Mariscal (1997) further studied acclimation and the process of
acquired protection of three selected species of anemone fishes against three selected
sea anemone species which were randomized to be natural host or unnatural host to
selective fish species. Only one fish species, A. clarkii was not stung by initial contact
to all sea anemone tentacles. Another species, A. ocellaris was stung by tentacles of
Heteractis crispa and Macrodactyla dorensis, as these two sea anemone were unnatural
host to A. ocellaris. For A. perideraion (Bleeker, 1855), adhesion and protection were
mixed from its natural and unnatural host species. Hence, to indicate that naive
anemone fish is innately protected can be misleading, as through experiments conducted
8
by many, including Miyagawa (1989) suggested that chemical cues enable the
symbiosis process. Miyagawa (1989) published the isolation of amphekuemin (1), a
pyridium alkaloid, that appealed swimming of A. perideraion fish towards its expected
sea anemone host, Radianthus kuekenthali.
(1)
It might be possible that more study on the variation of anemone fish species
mucous against selected host sea anemone species may provide knowledge to further
understand the protection mechanism and symbiosis adaptations.
2.1.1 False Clownfish
The False Clownfish, A. ocellaris (Cuvier in Cuvier and Valenciennes, 1830) is a
popularly photographed species of anemone fish in the coastal waters of peninsular
Malaysia and the coral triangle region. It can be found in shallow water reefs of 1-15
m depths waters because of their association to sea anemone which requires penetration
of sunlight for photosynthesis. It is easily recognised by its orange and white bands
with black linings on its small body and habitually swimming near or into the sea
anemone tentacles of the coral reefs (Timm, 2008).
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Figure 2.1. A photograph of a common false clownfish in Malaysian waters
contributed by Muhammad Faiz, Department of Marine Science, IIUM.
Its geographical distribution range from the tropical region that include countries
of Indo-West Pacific; Indian Ocean to Andaman and Nicobar Islands, Red Sea, South
East Asia region, Thailand, Malaysia, Indonesia, and the Philippines; and northwest
Australia to Singapore, and the Western Pacific to Taiwan and the Ryukyu Islands
(Ferrari Andrea and Ferrari Antonella, 2006). Their limited distribution is due to its
short larval stage resulting in limited larval dispersal.
Pacific
Ocean
South China
Sea
Indian
Ocean
Figure 2.2. Geographical distribution of false clownfish population. Retrieved
September 15, 2013, from FishBase website: http://www.fishbase.org/summary/6509.
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The false clownfish is categorised as a host specialist contrary to the Clark’s
anemone fish, A. clarkii which is categorised as a host generalist. Host specialist means
the fish, A. ocellaris only adapts to specific species of sea anemone, namely,
H. magnifica, S. gigantea, and S. mertensii. Host generalist fish like A. clarkii may live
mutually to almost all species of host sea anemone. The dependency of the fish to the
host might vary due to their swimming capabilities. Poor swimmers such as A. ocellaris
maintain close distance to its host while efficient swimmers may wonder far and retreat
quickly upon danger in a loose association manner.
The false clownfish adheres to strict linear dominance hierarchy, living in small
structure groups in each host sea anemone. They are sexually dimorphic with the size
of the female larger than the male fish. The largest fish in the hierarchy of one host sea
anemone is the dominating female fish and the second largest is the dominant male
which is also the breeder male in the group (Michael, 2008).
The distinct pair is monogamous throughout the lunar spawning period. The
dominant female will breed with only the dominant male fish by which the female
releases 100 to 1000 eggs fertilised by the male. Once hatched, the juvenile fill in the
ranks after the dominant male fish as non-breeder subordinates by size, largest to
smallest, which upon maturity or over crowdedness would find other host sea anemone
to inhabit.
As non-breeder subordinates, the juveniles or sub-adults demonstrate
psychophysical castration which the development of mature sex organ and growth are
repressed (Fautin & Allen, 1992).
2.2 SYMBIOSIS WITH SEA ANEMONE
Symbiosis is defined as the living of two organisms closely together or relying on one
another in a habitat or ecosystem for the benefit of both of the organisms. It differs from
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parasitism which only benefits one and harms another or commensalism which benefit
one party but does not help the other (Michael, 2008). In symbiotic relationship, both
organism will benefit mutually.
Mariscal (1970) reviewed reports of previous research observations to conclude
the nature of the anemone fish symbiosis to its host sea anemone. He denoted that both
benefit mutually by this relationship even though sea anemone may be observed
surviving in the wild without hosting fish. The fish are obligate symbiont that benefit
primarily by protection from predator, nesting and breeding grounds, parasite removal,
tactile stimulation and possibly eating the sea anemone as some food source.
On the other hand the sea anemone may benefit more as they also receive
protection from predator as the symbiont fish may help deter away and afford tactile
stimulation. As the fish swim around the tentacles, the fish may contribute to remove
parasites and necrotic tissues and organic and inorganic materials from the oral disc or
sea anemone along with providing the host with water and food circulation.
This is later proven as Szczebak et al. (2013) investigated on the anemonefish
movements and its effect on oxygenating the sea anemone host. Roopin, Henry and
Chadwick (2008) demonstrated nutrient transfer from the fish to the sea anemone.
Furthermore, studies found sea anemone with removed resident fish tend to die off.
This could also be due to predation of butterflyfish which come to graze on the polyps
of sea anemone when there are no anemonefish to chase them away.
Sea anemone are distributed worldwide having the greatest species diversity at
30 – 40 ° N and S. According to Fautin and Allen (1992), the 10 species of anemones
that host anemone fish are taxonomically diverse. These belong to five genera from
three families: family Stichodactylidae which includes S. haddoni, S. gigantea, S.
mertensii, H. magnifica, H. crispa, H. aurora, and H. malu; family Actiniidae species
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