HARMFUL MARINE DINOFLAGELLATES (Dinophyceu) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR

Tan Toh Hii

Master of Science 2012

I HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

TAN TOHHII

THESIS SUBMITIED IN FULFILMENT FOR THE DEGREE OF MASTER OF SCIENCE (MARINE BIOTECHNOLOGy)

INSTITUTE OF BIODIVERSITY AND ENVIRONMENTAL CONSERVATION (IDEC)

UNIVERSITI MALAYSIA SARA W AK

2012

DECLARATION

I hereby declare that the work in this thesis is my original work except for quotations

and summaries which have been duly acknowledged I also declare that it has not been

previously or concurrently submitted for any other degree at UNIMAS or other

institutions

TANTOHHII

09021505

Institute of Biodiversity and Environmental Conservation

Universiti Malaysia Sarawak

11

ACKNOWLEDGEMENTS

I would like to express my utmost and deepest gratitude and appreciation towards my

supervisors Dr Leaw Chui Pin and Dr Lim Po Teen who had made this dissertation a

success

I would also like to thank all those listed below who have assisted directly and

indirectly in data collections and data analyses

Members of the Malaysian Ecology and Oceanography of Harmful Algal Blooms

(MEOHAB) group Hartina Mohd Ali Lim Hong Chang Fareha Hilaluddin Siti

Zubaidah Kamarudin Teng Sing Tung Hii Kieng Soon Tuan Nurhariani Tuanshy

Halim Hii Kah Hung Chai Hui ling Yong Siok Chin Chow Luan lia and Kon Nyuk

Fung

Laboratory assistance and science officers Rahah bt Mohd Yakup Ting Woei Amin

Mangi Mohd Hasri AI-Hafiz b Haba and Nazri Latip

Collaborators Dr Yoshinobu Takata Roziawati binti Mohd Razali

Family and friends Tan Kok Meng Hii Ai Hung Tan Kok Kheng Hii Su Chu Lau

Chou Kuang Doreen Tan Robin Tan Lina Yek Ting Sie Yew Tung Lay Soon and

Hoe Yin Chen

This research project was funded by UNIMAS Small Grant Scheme and eScience

Fund to Dr Leaw Chui Pin

iii

HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

ABSTRACT

(Dinoflagellates are eukaryotic biflagellated unicellular organisms that can be found in

both freshwater and marine habitats Several species are harmful as they produce potent neurotoxins cause unusual blooming phenomena in the marine environment In Malaysia harmful algal blooms (HABs) event was first reported in 1976 Since then several studies on HABs have been undertaken focusing mainly in areas where poisoning cases were reported) In this study samplings were conducted in several locations including sites where no poisoning case has been reported but intensive aquaculture activity is ongoing Plankton and seaweed samples were collected and brought back to the laboratory Single-cell of dinoflagellates were isolated and clonal cultures established Both field and cultured specimens were observed under light and

scanning electron microscopy (SEM) Identification to species level was based on detailed investigation of the thecal plates Nucleotide sequences of the nuclearshyencoded ribosomal DNA were obtained from clonal cultures and preliminary toxin analysis was performed by ELISA and LC-MS A total of 46 taxa of harmful andor potentially harmful dinoflagellates were identified and documented from four locations (Cherating Pahang Port Dickson Negeri Sembi lan Tebrau Strait Johor and Kuching Sarawak) Two taxa are new records of occurrence in Malaysian waters namely Karlodinium veneficum from Tebrau Strait and Protoceratiumfukuyoii sp nov

from Semariang Notably the PST-producing dinoflagellates Alexandrium lamiyavanichii and Pyrodinium bahamense were found for the first time in Sarawak waters and the Straits of Malacca respectively Molecular data obtained from the present study and the molecular signatures identified for each species will serve as preliminary data for DNA barcoding of the harmful dinoflagellates Inventory of the harmful dinoflagellate species wiIl provide useful baseline infonnation of the harmful species present in Malaysian waters Furthermore distribution data on harmful dinoflagellates could be used to identify the hotspots of potential HABs incidences for early warning and HABs monitoring purposes

Key words harmful dinoflagellates Malaysia SEM Karlodinium veneficum

Protoceratiumfukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

IV

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

I HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

TAN TOHHII

THESIS SUBMITIED IN FULFILMENT FOR THE DEGREE OF MASTER OF SCIENCE (MARINE BIOTECHNOLOGy)

INSTITUTE OF BIODIVERSITY AND ENVIRONMENTAL CONSERVATION (IDEC)

UNIVERSITI MALAYSIA SARA W AK

2012

DECLARATION

I hereby declare that the work in this thesis is my original work except for quotations

and summaries which have been duly acknowledged I also declare that it has not been

previously or concurrently submitted for any other degree at UNIMAS or other

institutions

TANTOHHII

09021505

Institute of Biodiversity and Environmental Conservation

Universiti Malaysia Sarawak

11

ACKNOWLEDGEMENTS

I would like to express my utmost and deepest gratitude and appreciation towards my

supervisors Dr Leaw Chui Pin and Dr Lim Po Teen who had made this dissertation a

success

I would also like to thank all those listed below who have assisted directly and

indirectly in data collections and data analyses

Members of the Malaysian Ecology and Oceanography of Harmful Algal Blooms

(MEOHAB) group Hartina Mohd Ali Lim Hong Chang Fareha Hilaluddin Siti

Zubaidah Kamarudin Teng Sing Tung Hii Kieng Soon Tuan Nurhariani Tuanshy

Halim Hii Kah Hung Chai Hui ling Yong Siok Chin Chow Luan lia and Kon Nyuk

Fung

Laboratory assistance and science officers Rahah bt Mohd Yakup Ting Woei Amin

Mangi Mohd Hasri AI-Hafiz b Haba and Nazri Latip

Collaborators Dr Yoshinobu Takata Roziawati binti Mohd Razali

Family and friends Tan Kok Meng Hii Ai Hung Tan Kok Kheng Hii Su Chu Lau

Chou Kuang Doreen Tan Robin Tan Lina Yek Ting Sie Yew Tung Lay Soon and

Hoe Yin Chen

This research project was funded by UNIMAS Small Grant Scheme and eScience

Fund to Dr Leaw Chui Pin

iii

HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

ABSTRACT

(Dinoflagellates are eukaryotic biflagellated unicellular organisms that can be found in

both freshwater and marine habitats Several species are harmful as they produce potent neurotoxins cause unusual blooming phenomena in the marine environment In Malaysia harmful algal blooms (HABs) event was first reported in 1976 Since then several studies on HABs have been undertaken focusing mainly in areas where poisoning cases were reported) In this study samplings were conducted in several locations including sites where no poisoning case has been reported but intensive aquaculture activity is ongoing Plankton and seaweed samples were collected and brought back to the laboratory Single-cell of dinoflagellates were isolated and clonal cultures established Both field and cultured specimens were observed under light and

scanning electron microscopy (SEM) Identification to species level was based on detailed investigation of the thecal plates Nucleotide sequences of the nuclearshyencoded ribosomal DNA were obtained from clonal cultures and preliminary toxin analysis was performed by ELISA and LC-MS A total of 46 taxa of harmful andor potentially harmful dinoflagellates were identified and documented from four locations (Cherating Pahang Port Dickson Negeri Sembi lan Tebrau Strait Johor and Kuching Sarawak) Two taxa are new records of occurrence in Malaysian waters namely Karlodinium veneficum from Tebrau Strait and Protoceratiumfukuyoii sp nov

from Semariang Notably the PST-producing dinoflagellates Alexandrium lamiyavanichii and Pyrodinium bahamense were found for the first time in Sarawak waters and the Straits of Malacca respectively Molecular data obtained from the present study and the molecular signatures identified for each species will serve as preliminary data for DNA barcoding of the harmful dinoflagellates Inventory of the harmful dinoflagellate species wiIl provide useful baseline infonnation of the harmful species present in Malaysian waters Furthermore distribution data on harmful dinoflagellates could be used to identify the hotspots of potential HABs incidences for early warning and HABs monitoring purposes

Key words harmful dinoflagellates Malaysia SEM Karlodinium veneficum

Protoceratiumfukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

IV

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

DECLARATION

I hereby declare that the work in this thesis is my original work except for quotations

and summaries which have been duly acknowledged I also declare that it has not been

previously or concurrently submitted for any other degree at UNIMAS or other

institutions

TANTOHHII

09021505

Institute of Biodiversity and Environmental Conservation

Universiti Malaysia Sarawak

11

ACKNOWLEDGEMENTS

I would like to express my utmost and deepest gratitude and appreciation towards my

supervisors Dr Leaw Chui Pin and Dr Lim Po Teen who had made this dissertation a

success

I would also like to thank all those listed below who have assisted directly and

indirectly in data collections and data analyses

Members of the Malaysian Ecology and Oceanography of Harmful Algal Blooms

(MEOHAB) group Hartina Mohd Ali Lim Hong Chang Fareha Hilaluddin Siti

Zubaidah Kamarudin Teng Sing Tung Hii Kieng Soon Tuan Nurhariani Tuanshy

Halim Hii Kah Hung Chai Hui ling Yong Siok Chin Chow Luan lia and Kon Nyuk

Fung

Laboratory assistance and science officers Rahah bt Mohd Yakup Ting Woei Amin

Mangi Mohd Hasri AI-Hafiz b Haba and Nazri Latip

Collaborators Dr Yoshinobu Takata Roziawati binti Mohd Razali

Family and friends Tan Kok Meng Hii Ai Hung Tan Kok Kheng Hii Su Chu Lau

Chou Kuang Doreen Tan Robin Tan Lina Yek Ting Sie Yew Tung Lay Soon and

Hoe Yin Chen

This research project was funded by UNIMAS Small Grant Scheme and eScience

Fund to Dr Leaw Chui Pin

iii

HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

ABSTRACT

(Dinoflagellates are eukaryotic biflagellated unicellular organisms that can be found in

both freshwater and marine habitats Several species are harmful as they produce potent neurotoxins cause unusual blooming phenomena in the marine environment In Malaysia harmful algal blooms (HABs) event was first reported in 1976 Since then several studies on HABs have been undertaken focusing mainly in areas where poisoning cases were reported) In this study samplings were conducted in several locations including sites where no poisoning case has been reported but intensive aquaculture activity is ongoing Plankton and seaweed samples were collected and brought back to the laboratory Single-cell of dinoflagellates were isolated and clonal cultures established Both field and cultured specimens were observed under light and

scanning electron microscopy (SEM) Identification to species level was based on detailed investigation of the thecal plates Nucleotide sequences of the nuclearshyencoded ribosomal DNA were obtained from clonal cultures and preliminary toxin analysis was performed by ELISA and LC-MS A total of 46 taxa of harmful andor potentially harmful dinoflagellates were identified and documented from four locations (Cherating Pahang Port Dickson Negeri Sembi lan Tebrau Strait Johor and Kuching Sarawak) Two taxa are new records of occurrence in Malaysian waters namely Karlodinium veneficum from Tebrau Strait and Protoceratiumfukuyoii sp nov

from Semariang Notably the PST-producing dinoflagellates Alexandrium lamiyavanichii and Pyrodinium bahamense were found for the first time in Sarawak waters and the Straits of Malacca respectively Molecular data obtained from the present study and the molecular signatures identified for each species will serve as preliminary data for DNA barcoding of the harmful dinoflagellates Inventory of the harmful dinoflagellate species wiIl provide useful baseline infonnation of the harmful species present in Malaysian waters Furthermore distribution data on harmful dinoflagellates could be used to identify the hotspots of potential HABs incidences for early warning and HABs monitoring purposes

Key words harmful dinoflagellates Malaysia SEM Karlodinium veneficum

Protoceratiumfukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

IV

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

ACKNOWLEDGEMENTS

I would like to express my utmost and deepest gratitude and appreciation towards my

supervisors Dr Leaw Chui Pin and Dr Lim Po Teen who had made this dissertation a

success

I would also like to thank all those listed below who have assisted directly and

indirectly in data collections and data analyses

Members of the Malaysian Ecology and Oceanography of Harmful Algal Blooms

(MEOHAB) group Hartina Mohd Ali Lim Hong Chang Fareha Hilaluddin Siti

Zubaidah Kamarudin Teng Sing Tung Hii Kieng Soon Tuan Nurhariani Tuanshy

Halim Hii Kah Hung Chai Hui ling Yong Siok Chin Chow Luan lia and Kon Nyuk

Fung

Laboratory assistance and science officers Rahah bt Mohd Yakup Ting Woei Amin

Mangi Mohd Hasri AI-Hafiz b Haba and Nazri Latip

Collaborators Dr Yoshinobu Takata Roziawati binti Mohd Razali

Family and friends Tan Kok Meng Hii Ai Hung Tan Kok Kheng Hii Su Chu Lau

Chou Kuang Doreen Tan Robin Tan Lina Yek Ting Sie Yew Tung Lay Soon and

Hoe Yin Chen

This research project was funded by UNIMAS Small Grant Scheme and eScience

Fund to Dr Leaw Chui Pin

iii

HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

ABSTRACT

(Dinoflagellates are eukaryotic biflagellated unicellular organisms that can be found in

both freshwater and marine habitats Several species are harmful as they produce potent neurotoxins cause unusual blooming phenomena in the marine environment In Malaysia harmful algal blooms (HABs) event was first reported in 1976 Since then several studies on HABs have been undertaken focusing mainly in areas where poisoning cases were reported) In this study samplings were conducted in several locations including sites where no poisoning case has been reported but intensive aquaculture activity is ongoing Plankton and seaweed samples were collected and brought back to the laboratory Single-cell of dinoflagellates were isolated and clonal cultures established Both field and cultured specimens were observed under light and

scanning electron microscopy (SEM) Identification to species level was based on detailed investigation of the thecal plates Nucleotide sequences of the nuclearshyencoded ribosomal DNA were obtained from clonal cultures and preliminary toxin analysis was performed by ELISA and LC-MS A total of 46 taxa of harmful andor potentially harmful dinoflagellates were identified and documented from four locations (Cherating Pahang Port Dickson Negeri Sembi lan Tebrau Strait Johor and Kuching Sarawak) Two taxa are new records of occurrence in Malaysian waters namely Karlodinium veneficum from Tebrau Strait and Protoceratiumfukuyoii sp nov

from Semariang Notably the PST-producing dinoflagellates Alexandrium lamiyavanichii and Pyrodinium bahamense were found for the first time in Sarawak waters and the Straits of Malacca respectively Molecular data obtained from the present study and the molecular signatures identified for each species will serve as preliminary data for DNA barcoding of the harmful dinoflagellates Inventory of the harmful dinoflagellate species wiIl provide useful baseline infonnation of the harmful species present in Malaysian waters Furthermore distribution data on harmful dinoflagellates could be used to identify the hotspots of potential HABs incidences for early warning and HABs monitoring purposes

Key words harmful dinoflagellates Malaysia SEM Karlodinium veneficum

Protoceratiumfukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

IV

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

HARMFUL MARINE DINOFLAGELLATES (DINOPHYCEAE) OF MALAYSIA INCLUDING NEW RECORDS OF TAXA BASED ON

MORPHOLOGY AND MOLECULAR CHARACTERIZATION

ABSTRACT

(Dinoflagellates are eukaryotic biflagellated unicellular organisms that can be found in

both freshwater and marine habitats Several species are harmful as they produce potent neurotoxins cause unusual blooming phenomena in the marine environment In Malaysia harmful algal blooms (HABs) event was first reported in 1976 Since then several studies on HABs have been undertaken focusing mainly in areas where poisoning cases were reported) In this study samplings were conducted in several locations including sites where no poisoning case has been reported but intensive aquaculture activity is ongoing Plankton and seaweed samples were collected and brought back to the laboratory Single-cell of dinoflagellates were isolated and clonal cultures established Both field and cultured specimens were observed under light and

scanning electron microscopy (SEM) Identification to species level was based on detailed investigation of the thecal plates Nucleotide sequences of the nuclearshyencoded ribosomal DNA were obtained from clonal cultures and preliminary toxin analysis was performed by ELISA and LC-MS A total of 46 taxa of harmful andor potentially harmful dinoflagellates were identified and documented from four locations (Cherating Pahang Port Dickson Negeri Sembi lan Tebrau Strait Johor and Kuching Sarawak) Two taxa are new records of occurrence in Malaysian waters namely Karlodinium veneficum from Tebrau Strait and Protoceratiumfukuyoii sp nov

from Semariang Notably the PST-producing dinoflagellates Alexandrium lamiyavanichii and Pyrodinium bahamense were found for the first time in Sarawak waters and the Straits of Malacca respectively Molecular data obtained from the present study and the molecular signatures identified for each species will serve as preliminary data for DNA barcoding of the harmful dinoflagellates Inventory of the harmful dinoflagellate species wiIl provide useful baseline infonnation of the harmful species present in Malaysian waters Furthermore distribution data on harmful dinoflagellates could be used to identify the hotspots of potential HABs incidences for early warning and HABs monitoring purposes

Key words harmful dinoflagellates Malaysia SEM Karlodinium veneficum

Protoceratiumfukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

IV

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

DINOFLAGELAT MARIN BERBAHAY A (DINOPHYCEAE) DI MALAYSIA TERMASUK REKOD TAKSA BARU BERDASARKAN MORFOLOGI DAN

PENCIRIAN MOLEKUL

ABSTRAK

Dinojlagelat ialah organism eukariot dwi jlagella unisel yang boleh dijumpai di habitat air tawar dan laut Dalam persekitaran marinterdapat beberapa spesis yang merbahaya di mana mereka menghasilkan neurotoksin yang merbahaya Sesetengah menyebabkan fenomena ledakan yang luar biasa Di Malaysia peristiwa ledakan alga berbahaya (HAB) telah dilaporkan sejak tahun 1976 Sejak itu kajian HAB telah dijalankan Walau bagaimanapun kajian HAB di Malaysia dijalankan secara pasif berdasarkan tempat-tempat di mana kes-kes keracunan dilaporkan Dalam kajian ini persampelan telah dijalankan di lokasi yang terpilih di perairan Malaysia Kawasan persampelan yang dipilih adalah tempat yang mempunyai aktiviti akuakultur tetapi tiada laporan kes keracunan seta kat ini Sampel plankton dan rumpai laut telah dikumpul dan dibawa balik ke makma Sel tunggal dinojlagelat diasing dan kultur don dikembangkan Morfologi spesimen lapangan dan kultur telah diperhatikan dengan menggunakan mikroskop cahaya dan mikroskop pengimbasan elektron (SEM) Pengenalan kepada peringkat spesies adalah berdasarkan penyiasatan terperinci kepingan theka Jujukan nukleotida DNA nucleus berkod ribosomal telah diperolehi daripada kultur klon dan analisa toksin telah dijalankan dengan ELISA dan LC-MS Sejumlah 46 takiO dinojlagelat berbahaya atau berpotensi membahayakan telah dikenalpasti dan didokumenkan dari empat lokasi (Cherating Pahang Port Dickson Negeri Sembian Selat Tebrau Johor dan Kuching Sarawak) Dua taksa rekod baru di perairan Malaysia ialah Karlodinium veneficum

dari Selat Tebrau dan Protoceratium fukuyoii sp nov dari Semariang Dinojlagelat penghasilan toxin PSP Alexandrium tamiyavanichii dan Pyrodinium bahamense

telah dijumpai untuk kali pertama di perairan Sarawak dan Selat Melaka masingshymasing Data molekul yang diperolehi daripada kajian ini dan pengenalan molekul yang dikenalpasti bagi setiap spesies akan digunakan sebagai data awal untuk barkod DNA dinojlagelat berbahaya Inventori spesies dinojlagelat berbahaya berupaya menyediakan maklumat asas tentang spesies berbahaya yang didapati di perairan Malaysia Tambahan pula data taburan dinojlagelat berbahaya boleh digunakan unluk mengenalpasti lokasi berpotensi kejadian HAB untuk amaran awal dan tujuan pemantauan HAB

Kata Kunci Dinojlagellat Malaysia SEM Karlodinium veneficum Protoceratium

fukuyoii sp nov Alexandrium tamiyavanichii Pyrodinium bahamense

v

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

Pusat Khidmat Maldumat AkaOemik UNIVERSnl MALAYSIA SARAWA)(

DECLARATION TABLE OF CONTENTS Page

ii ACKNOWLEDGEMENTS iii ABSTRACT IV

ABSTRAK V

TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x

CHAPTER I INTRODUCTION 1

11 Dinoflagellates and red tides 1

12 History of HABs in Malaysia 2

13 Paralytic Shellfish Poisoning (PSP) 7 131 The causative organisms 7 132 Saxitoxins (STX) 10

14 Diarrhetic Shellfish Poisoning (DSP) 10 141 The causative organisms 11 142 Okadaic acid (OA) pectenotoxins (PTX) and yessotoxins 12

(YTX)

15 Ciguatera Fish Poisoning (CFP) 13 151 The causative organisms 14 152 Ciguatoxins maitotoxins gambiertoxins 15

16 Justification of the study 16

17 Objectives of the study 17

CHAPTER II DINOFLAGELLATE ASSEMBLAGES IN 18 SELECTED AREAS OF MALAYSIAN WATERS

21 Introduction 18

22 Materials and methods 20

221 Sample collection 20 222 Species identification 22

2221 Light microscopy 22 2222 Scanning electron microscopy 22

23 Results and Discussions 24 231 Dinoflagellates assemblages 24 232 HABs species inventory in Malaysia 27

VI

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

Page 233 Species description 32

2331 Akashiwo sanguinea (K Hirasaka) G Hansen amp 32 o Moestrup 2000

2332 Alexandrium affine (Inoue and Fukuyo) Balech 34 1985

2333 Alexandrium tamiyavanichii Balech 1994 36 2334 Balechina sp 37 2335 Cochlodinium convolutum Kofoid amp Swezy 1921 38 2336 Coolia malayensis CP Leaw PT Lim and G 39

Usup2010 2337 Dinophysis acuminata ClaparMe and Lachmann 41

1859 2338 Dinophysis caudata Saville-Kent 1881 43 2339 Dinophysis miles Cleve 1900 45 23310 Dinophysis norvegica Claparede at Lachmann 47

1859

23311 Diplopsais lenticula Bergh 1881 48 23312 Diplopsalopsis orbicularis (Paulsen) Meunier 50

1910 23313 Gonyaulax cf scrippsae Kofoid 1911 51 23314 Gonyaulax polygramma Stein 1883 52 23315 Gonyaulax spinijera (Claparede et Lachmann) 54

Diesing 1866 23316 Gymnodinium instriatum Freudenthal amp Lee 55

1963 23317 Gyrodinium spirale Bergholtz et aI 2006 Kofoid 57

et Swezy 1921 23318 Karenia mikimotoi Miyake et Kominami ex Oda 59

1935 23319 Karlodinium veneficum (D Ballantine) J Larsen 61

2000 23320 Lingulodinium polyedrum (Stein) Dodge 1989 66 23321 Neoceratium furca (Ehrenberg) ClaparMe and 67

Lachmann 1859 23322 Noctiluca scintillans (Macartney) Kofoid et 69

Swezy 1920 233 23 Prorocentrum gracile SchUtt 1895 70 23324 Prorocentrum micans Ehrenberg 1834 72 23325 Prorocentrum rhathymum Loeblich Sherley amp 73

Schmidt 1979 23326 Protoceratium reticulatum (Claparede amp 75

Lachmann) BUtschli 1885 23327 Protoceratium sp 1 77

23328 Protoperidinium latissimum Sommer et al Balech 79 1974

23329 Protoperidinium marukawai (Abe) Balech 1974 80

vii

Page 23330 Protoperidinium pyriforme (Paulsen) Balech 1974 81 23331 Peridinium quinquecorne (Abe) Balech 1974 82 23332 Protoperidinium sp I 83 23333 Protoperidinium sp2 84 23334 Protoperidinium subinerme (Paulsen) Loeblich III 85

1970 23335 Pyrodinium bahamense var compressum (Bohm) 86

Steidinger Tester et Taylor 1980 23336 Pyrophacus steinii (Schiller) Wall amp Dal 1971 87 23337 Scrippsiella trochoidea (Stein) Balech ex 89

Loeblich III 1965

24 Conclusion 90

CHAPTER III DETAILED MORPHOLOGY AND MOLECULAR 92 CHARACTERIZATION OF HARMFUL ANDOR POTENTIALLY HARMFUL DINOFLAGELLATES IN MALAYSIAN WATERS

31 Introduction 92

32 Materials and methods 95

321 Algal cultures 95 322 Morphological observation 95 323 Molecular analyses 97

3231

3232 3233 3234 3235

Total genomic DNA isolation gene amplification 97 and sequencing Sequences analysis and taxon sampling 98 Secondary structure prediction 98 Analyses of genetic diversity 98 Phylogenetic analyses 99

33 Results and Discussion 100

331 Coolia malayensis from Lundu the southern of Malaysian 100 Borneo

332 Gambierdiscus belizeanus from east coast of Sabah 117 333 Prorocentrum lima from Sabah east coast 122 334 Prorocentrum rhathymum from Semariang Sarawak 126 335 Description of a new species Protoceratium fukuyoii sp 133

nov from Semariang Sarawak 336 Alexandrium tamiyavanichii from Semariang Sarawak 141

CHAPTER IV CONCLUSION 150

REFERENCES 152 APPENDICES 169

viii

LIST OF TABLE Table Page 11 Historical event of HABs in Malaysia from 1976 - 2010 6 21 GPS coordinates of sampling sites and sampling date in this study 20 22 List of dinoflagellates found at each sampling sites from July 2009 to 26

Nov 2010 Potentially harmful or harmful dinoflagellates are in bold

23 Number of HABs dinoflagellates species found in this study 27 24 Species inventory of harmful dinoflagellates species in Malaysia 28

their impact and locations References in asterisks are referring to the impacts

25 Morphological comparison of species in the genus of Karlodinium 64 Morphological characteristics of each species were compiled from

literatures (Bergholtz et aI 2006 Daugbjerg 2000 De Salas et aI 2005 De Salas et aI 2008 Siano et aI 2009)

31 Cultures used in this study with species strain label sampling dates 96 locations and isolators

32 List of primers selected for PCR amplification of LSU rONA and ITS 97 region

33 PCR amplification condition for LSU rONA and ITS 98 34 Morphometric measurements of Coolia malayensis from Lundu 104

Sarawak with comparison to the other Coolia species described Cell size estimated as mean length (L) width (W) and ratio of Land W (LW) Po apical pore plate Number of cells observed n

35 LSU nucleotide sequence obtained in this study and sequences 106 retrieved from Genbank

36 Compensatory base changes (CBCs) found in pairwise comparison of 110 ITS2 transcripts of Coolia malayensis and C monolis Strains in boldface are strains obtained in this study

37 The ITS haplotypes of Malaysian Coolia malayensis populations in 113 the ITS region of rONA Nucleotide differences at the same sequence

position are shown for each haplotype The geographical origin of

each haplotype is listed

38 Morphological measurements of Gambierdiscus GdSA03 from Kota 121 Kinabalu Sabah with comparison to other closely related Gambierdiscus species Number of cells observed n = 30 cells

Values in parentheses are the mean sizes

39 Species list of Prorocenlrum species and their LSU rONA Genbank 131 accessions used in this study

310 Nucleotide sequence obtained from this study and sequences retrieved 148 from Genbank used in the phylogenetic analyses

ix

LIST OF FIGURES

Figure Page 11 Scanning micrographs of Pyrodinium bahamense var compressum 8

from Kota Kinabalu Sabah (source Lim I 999Leaw et aI 2005)

12 Thecal plate arrangement ofAlexandrium species (source Taylor 9 1995)

21 Map of Malaysia showing the sampling sites in this study 21 22 Light micrograph of Akashiwo sanguinea from Semariang Sarawak 33

(A) A vegetative cell showing light green chloroplast and a longitudinal flagellum (arrow) (B) Auto-fluorescence showing chloroplast content Scale bar = 10 11m

23 Light and electron micrographs of Alexandrium affine from 35 Semariang Sarawak (A) A chain of four cells (B) Epiflourescence of the cell showing the arrangement of chloroplast Scale bar = 10 11m (C-F) SEM (C) Lateral view of the cell (D) Apical view of a crushed cell (E) Shrunk cell showing apical pore (F) Ventrashyantapical view showing the sulcus list (sl)

24 Light micrographs of Alexandrium tamiyavanichii from Semariang 36 Sarawak (A) A chain of two vegetative cells (B) Red fluorescence showing chloroplast arrangement Scale bar = 100 11m

25 A chain of four cells Balechina sp from Kota Kinabalu Sabah Scale 37 bar = 10 11m

26 Light micrographs of Cochlodinium convolutum from Semariang 38 Sarawak (A) Light brown-greenish pigmented cells (B) Red

autofluorescence of the cell showing chloroplast content Scale bar = 101lm

27 Scanning electron micrographs of Cooia malayensis from Lundu 40 Sarawak (A) Lateral view showing oval shape (B) Antapical-dorsal view showing the largest 3 plate (C-D) Ventral view (E) Dorsal view where cell appear to be round (F) Lateral view Scale bar=5Ilm

28 Dinophysis acuminata from Tebrau Strait Johor (A) Lateral view 42 (B) Ventral view (C) Dorsal-antapical view Scale bar = 10 11m

29 Light and scanning electron micrographs of Dinophysis caudata from 44 Santubong Sarawak (A-B) SEM Scale bar = 20 11m (C-F) LM showing chloroplast content

210 Light micrographs of Dinophysis miles from Santubong Sarawak 46 (A) Cell with golden brown chloroplast content Left sulcal list (LSL) eveident (B) Cell without chloroplast content (C) Three ribs supporting the LSL

2 11 Light micrographs of Dinophysis norvegica from Semariang 47 Sarawak (A) Cell is pointed at the antapex (arrow) (B) Red autofluorescence showing the chloroplast arrangement Scale bar = 100 11m

x

Figure Page 212 Scanning electron micrographs of Diplosalis lenticula from Tebrau 49

Strait Johor (A) Apical view showing the apical pore (ap) (B) Antapical view (C) Dorsal view with the sulcus

2l3 Scanning electron micrographs of Diplopsalopsis orbicularis covered 50 in dirt Scale bar = 10 )tm

214 Scanning electron micrographs of Gonyaulax cf scrippsae from 51 Cherating Pahang (A) Lateral view of cell with large trichocyst pores (B) Anterio-ventral view of cell showing two cingulum displacement and sulcus (s)

215 Scanning electron micrograph of Gonyaulax polygramma from 53 Cherating Pahang

216 Light micrographs of Gonyaulax spinifera from Semariang Sarawak 54 (A) Cell with apical horn (arrow) (B) Red autofluorescence showing chloroplast content Scale bar = 10 )tm

217 Scanning electron micrographs of Gymnodinium instriatum from 56 Santubong Sarawak (A) Ventral view showing the cingulum displacement (B) Dorsal view showing slightly bilobed hypocone and conical epicone (C amp D) Antapical view showing the sulcal intrusion into the antapex Scale bar = 5 )tm

218 Scanning electron micrograph of Gyrodinium spirale from Tebrau 58 Strait Johor Scale bar = 20 )tm

219 Scanning electron micrograph of Gymnodinium mikimotoi from 60 Tebrau Strait Johor showing vertical apical groove (ag)

220 Scanning electron micrographs of Karlodinium veneficum from 63 Tebrau Strait Johor (A) Apical-dorsal view showing elongated ventral pore (vp) and apical groove (ag) (B) Dorsal view showing clearly the 3x cingulum displacement (cd) descending to the right (C) Ventral view showing the starting of apical groove Scale bar = 5 )tm

221 Scanning electron micrographs of Lingulodinium polyedrum from 66 Clterating Pahang

222 Light and scanning electron micrographs of Neocertium furca from 68 Tebrau Strait Johor (A) SEM showing the cell and the longitudinal ridges (B-C) LM Cells from Semariang Sarawak (B) A chain of two vegetative cells (C) Red autofluorescence showing chloroplast content Scale bar = 20 )tm

223 Light micrographs of Noctiluca scintillans from Santubong Sarawak 69 (A) Cells with greenish spots (B) Apical view (C) Antapical view showing flagellum

224 Light and scanning electron micrographs of Prorocentrum gracile 71 from Semariang Sarawak (A-B) LM (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-D) SEM Scale bar = 10 )tm

XI

Figure Page 225 Scanning electron micrograph of Prorocentrum micans from Tebrau 73

Strait lohor

226 (A) Light micrograph of Prorocentrum rhathymum showing short 74 apical spine (arrow head) (B) Red auto florescence showing chloroplast content (C-F) Epiflorescence micrograph (C) Right valve (D) Left valve (E) Periflagellar area (arrow head) (F)

Intercalary band (arrow) Scale bar = 10 pm

227 Scanning electron micrographs of Protoceratium reticulatum from 76 Cherating Pahang (A) Lateral view Cingulum displacement (arrow) (B) Dorsal-antapical view

228 Scanning electron micrograph of a new morphotype Protoceratium 78 sp I from Cherating Pahang showing the ventral-antapical view

229 Scanning electron micrograph of Protoperidinium latissimum from 79 Cherating Pahang Ventral view

230 Scanning electron micrographs of Protoperidinium marukawai from 80 Cherating Pahang Scale bar = 10 urn

231 Scanning electron micrographs of Protoperidinium pyriforme from 81 Cherating Pahang (A) Dorso-antapical view showing two antapical

spines (B) Dorsal view Apical horn is present (arrowhead) Scale bar = 10 pm

232 Scanning electron micrograph of Peridinium quinquecorne from 82 Cherating Pahang Ventral view

233 Scanning electron micrograph of Protoperidinium sp I from 83 Cherating Pahang Apical ventral view

234 Scanning electron micrograph of Protoperidinium sp 2 from 84 Kampung Geliga Besar Terengganu Dorsal view One of the antapical spine is covered in dirt

235 Scanning electron micrograph of Protoperidinium subinerme from 85 Cherating Pahang Apical view

236 Light micrograph of Pyrodinium bahamense var compressum from 86 Port Dickson Negeri Sembilan

237 Light micrographs of Pyrophacus stein from Semariang Sarawak 88 (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-H) Calcoflour stained ceUs showing thecal plates

238 Scanning electron micrograph of Scrippsiella trochoidea from Tebrau 89 Strait Johor Scale bar = 10 pm

XII

Figure Page 31 Clonal culture of Coolia malayensis showing the string of mucilage 100

(arrow) formed at the surface of the medium

32 Scanning electron microscopy of Coolia malayensis from Lundu 103 Sarawak (A) Apical view showing the large 6 (B) Dorsal view showing apical pore Po (C) Apical-ventral view showing I and 7 plates (0) Ventral view showing sulcus (E-F) Antapical-ventral views showing the large 3 (G) Apical pore complex showing long silt of apical pore (H) Minute perforations were observed within the surface pores (I) Lateral view of the cell

33 Phylogenetic tree of Coolia species derived from Bayesian analysis 107 of LSU rONA sequences The C malayensis strains from Lundu Sarawak used in this study are in boldface MP and ML bootstrap and BI posterior probability values are shown at the left of the internal nodes (MPIMLIBI) The four end-clades were labeled as Clade I 11 III and IV for comparison Scale bar =01 substitution per site

34 Secondary structural diagram of Coolia malayensis (CmLDO I) ITS2 109 transcript from Lundu Sarawak with universal motif shown AAA and GUU motif showing sequence conservation for 14 ITS2 sequences analyzed Arrow indicates U-U mismatch

35 Minimum spanning network of ITS haplotypes found in Malaysian 112 Coolia malayensis populations

36 Profile-neighbor joining (PNJ) tree of Coolia species Lundu strains 114 of C malayensis are in boldface

37 Gambierdiscus belizeanus GdSA03 from Kota Kinabalu Sabah (A) 119 LM Lugols stained cell showing the short longitudinal flagellum (arrow) (B) Epifluorescent micrographs of divided cells showing both epi- and hypotheca (C) Ventral view of cell showing first precingular plate (I ) and trapezoid 7 immersed into the ascending cingUlum and the deep sulcus Note the striation of the intercalary bands and the ridged margin of cingulum list (0) Apical view of cel l (E-F) antapical view of cell I p is small and narrow (G) Close view of the apical pore complex (APC) (H) Close view of the sulcal region showing the Sda t Sdp Ssp and Ssa plates

38 Prorocentrum lima strain PLKdO 1 from Kudat Sabah (A) Culture in 122 flask Clump of cells floating in the water column due to bubbles produced during photosynthesis activity (B) P lima cell observed under LM with 200x magnification Pyrenoid is located in the center of the cell (arrow)

39 Prorocentrum lima from Kudat Sabah SEM (A) valve views 125 showing two cells with left and right valves (B) Left valve view of the cell showing the absence of pores at the pyrenoid region (C) Right valve view of the cell showing the absence of pores at the pyrenoid region (0) Side view of the cell Arrow showing marginal pores along the circumference of the cell (E) Prominent notch with the eight plates labeled accordingly (F) The apical pore region where the flagellar pore f is larger than the auxiliary pore a

310 Cells ofProrocentrum rhathymum in culture tube showing planktonic 126 cells

Xlll

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

LIST OF FIGURES

Figure Page 11 Scanning micrographs of Pyrodinium bahamense var compressum 8

from Kota Kinabalu Sabah (source Lim I 999Leaw et aI 2005)

12 Thecal plate arrangement ofAlexandrium species (source Taylor 9 1995)

21 Map of Malaysia showing the sampling sites in this study 21 22 Light micrograph of Akashiwo sanguinea from Semariang Sarawak 33

(A) A vegetative cell showing light green chloroplast and a longitudinal flagellum (arrow) (B) Auto-fluorescence showing chloroplast content Scale bar = 10 11m

23 Light and electron micrographs of Alexandrium affine from 35 Semariang Sarawak (A) A chain of four cells (B) Epiflourescence of the cell showing the arrangement of chloroplast Scale bar = 10 11m (C-F) SEM (C) Lateral view of the cell (D) Apical view of a crushed cell (E) Shrunk cell showing apical pore (F) Ventrashyantapical view showing the sulcus list (sl)

24 Light micrographs of Alexandrium tamiyavanichii from Semariang 36 Sarawak (A) A chain of two vegetative cells (B) Red fluorescence showing chloroplast arrangement Scale bar = 100 11m

25 A chain of four cells Balechina sp from Kota Kinabalu Sabah Scale 37 bar = 10 11m

26 Light micrographs of Cochlodinium convolutum from Semariang 38 Sarawak (A) Light brown-greenish pigmented cells (B) Red

autofluorescence of the cell showing chloroplast content Scale bar = 101lm

27 Scanning electron micrographs of Cooia malayensis from Lundu 40 Sarawak (A) Lateral view showing oval shape (B) Antapical-dorsal view showing the largest 3 plate (C-D) Ventral view (E) Dorsal view where cell appear to be round (F) Lateral view Scale bar=5Ilm

28 Dinophysis acuminata from Tebrau Strait Johor (A) Lateral view 42 (B) Ventral view (C) Dorsal-antapical view Scale bar = 10 11m

29 Light and scanning electron micrographs of Dinophysis caudata from 44 Santubong Sarawak (A-B) SEM Scale bar = 20 11m (C-F) LM showing chloroplast content

210 Light micrographs of Dinophysis miles from Santubong Sarawak 46 (A) Cell with golden brown chloroplast content Left sulcal list (LSL) eveident (B) Cell without chloroplast content (C) Three ribs supporting the LSL

2 11 Light micrographs of Dinophysis norvegica from Semariang 47 Sarawak (A) Cell is pointed at the antapex (arrow) (B) Red autofluorescence showing the chloroplast arrangement Scale bar = 100 11m

x

Figure Page 212 Scanning electron micrographs of Diplosalis lenticula from Tebrau 49

Strait Johor (A) Apical view showing the apical pore (ap) (B) Antapical view (C) Dorsal view with the sulcus

2l3 Scanning electron micrographs of Diplopsalopsis orbicularis covered 50 in dirt Scale bar = 10 )tm

214 Scanning electron micrographs of Gonyaulax cf scrippsae from 51 Cherating Pahang (A) Lateral view of cell with large trichocyst pores (B) Anterio-ventral view of cell showing two cingulum displacement and sulcus (s)

215 Scanning electron micrograph of Gonyaulax polygramma from 53 Cherating Pahang

216 Light micrographs of Gonyaulax spinifera from Semariang Sarawak 54 (A) Cell with apical horn (arrow) (B) Red autofluorescence showing chloroplast content Scale bar = 10 )tm

217 Scanning electron micrographs of Gymnodinium instriatum from 56 Santubong Sarawak (A) Ventral view showing the cingulum displacement (B) Dorsal view showing slightly bilobed hypocone and conical epicone (C amp D) Antapical view showing the sulcal intrusion into the antapex Scale bar = 5 )tm

218 Scanning electron micrograph of Gyrodinium spirale from Tebrau 58 Strait Johor Scale bar = 20 )tm

219 Scanning electron micrograph of Gymnodinium mikimotoi from 60 Tebrau Strait Johor showing vertical apical groove (ag)

220 Scanning electron micrographs of Karlodinium veneficum from 63 Tebrau Strait Johor (A) Apical-dorsal view showing elongated ventral pore (vp) and apical groove (ag) (B) Dorsal view showing clearly the 3x cingulum displacement (cd) descending to the right (C) Ventral view showing the starting of apical groove Scale bar = 5 )tm

221 Scanning electron micrographs of Lingulodinium polyedrum from 66 Clterating Pahang

222 Light and scanning electron micrographs of Neocertium furca from 68 Tebrau Strait Johor (A) SEM showing the cell and the longitudinal ridges (B-C) LM Cells from Semariang Sarawak (B) A chain of two vegetative cells (C) Red autofluorescence showing chloroplast content Scale bar = 20 )tm

223 Light micrographs of Noctiluca scintillans from Santubong Sarawak 69 (A) Cells with greenish spots (B) Apical view (C) Antapical view showing flagellum

224 Light and scanning electron micrographs of Prorocentrum gracile 71 from Semariang Sarawak (A-B) LM (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-D) SEM Scale bar = 10 )tm

XI

Figure Page 225 Scanning electron micrograph of Prorocentrum micans from Tebrau 73

Strait lohor

226 (A) Light micrograph of Prorocentrum rhathymum showing short 74 apical spine (arrow head) (B) Red auto florescence showing chloroplast content (C-F) Epiflorescence micrograph (C) Right valve (D) Left valve (E) Periflagellar area (arrow head) (F)

Intercalary band (arrow) Scale bar = 10 pm

227 Scanning electron micrographs of Protoceratium reticulatum from 76 Cherating Pahang (A) Lateral view Cingulum displacement (arrow) (B) Dorsal-antapical view

228 Scanning electron micrograph of a new morphotype Protoceratium 78 sp I from Cherating Pahang showing the ventral-antapical view

229 Scanning electron micrograph of Protoperidinium latissimum from 79 Cherating Pahang Ventral view

230 Scanning electron micrographs of Protoperidinium marukawai from 80 Cherating Pahang Scale bar = 10 urn

231 Scanning electron micrographs of Protoperidinium pyriforme from 81 Cherating Pahang (A) Dorso-antapical view showing two antapical

spines (B) Dorsal view Apical horn is present (arrowhead) Scale bar = 10 pm

232 Scanning electron micrograph of Peridinium quinquecorne from 82 Cherating Pahang Ventral view

233 Scanning electron micrograph of Protoperidinium sp I from 83 Cherating Pahang Apical ventral view

234 Scanning electron micrograph of Protoperidinium sp 2 from 84 Kampung Geliga Besar Terengganu Dorsal view One of the antapical spine is covered in dirt

235 Scanning electron micrograph of Protoperidinium subinerme from 85 Cherating Pahang Apical view

236 Light micrograph of Pyrodinium bahamense var compressum from 86 Port Dickson Negeri Sembilan

237 Light micrographs of Pyrophacus stein from Semariang Sarawak 88 (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-H) Calcoflour stained ceUs showing thecal plates

238 Scanning electron micrograph of Scrippsiella trochoidea from Tebrau 89 Strait Johor Scale bar = 10 pm

XII

Figure Page 31 Clonal culture of Coolia malayensis showing the string of mucilage 100

(arrow) formed at the surface of the medium

32 Scanning electron microscopy of Coolia malayensis from Lundu 103 Sarawak (A) Apical view showing the large 6 (B) Dorsal view showing apical pore Po (C) Apical-ventral view showing I and 7 plates (0) Ventral view showing sulcus (E-F) Antapical-ventral views showing the large 3 (G) Apical pore complex showing long silt of apical pore (H) Minute perforations were observed within the surface pores (I) Lateral view of the cell

33 Phylogenetic tree of Coolia species derived from Bayesian analysis 107 of LSU rONA sequences The C malayensis strains from Lundu Sarawak used in this study are in boldface MP and ML bootstrap and BI posterior probability values are shown at the left of the internal nodes (MPIMLIBI) The four end-clades were labeled as Clade I 11 III and IV for comparison Scale bar =01 substitution per site

34 Secondary structural diagram of Coolia malayensis (CmLDO I) ITS2 109 transcript from Lundu Sarawak with universal motif shown AAA and GUU motif showing sequence conservation for 14 ITS2 sequences analyzed Arrow indicates U-U mismatch

35 Minimum spanning network of ITS haplotypes found in Malaysian 112 Coolia malayensis populations

36 Profile-neighbor joining (PNJ) tree of Coolia species Lundu strains 114 of C malayensis are in boldface

37 Gambierdiscus belizeanus GdSA03 from Kota Kinabalu Sabah (A) 119 LM Lugols stained cell showing the short longitudinal flagellum (arrow) (B) Epifluorescent micrographs of divided cells showing both epi- and hypotheca (C) Ventral view of cell showing first precingular plate (I ) and trapezoid 7 immersed into the ascending cingUlum and the deep sulcus Note the striation of the intercalary bands and the ridged margin of cingulum list (0) Apical view of cel l (E-F) antapical view of cell I p is small and narrow (G) Close view of the apical pore complex (APC) (H) Close view of the sulcal region showing the Sda t Sdp Ssp and Ssa plates

38 Prorocentrum lima strain PLKdO 1 from Kudat Sabah (A) Culture in 122 flask Clump of cells floating in the water column due to bubbles produced during photosynthesis activity (B) P lima cell observed under LM with 200x magnification Pyrenoid is located in the center of the cell (arrow)

39 Prorocentrum lima from Kudat Sabah SEM (A) valve views 125 showing two cells with left and right valves (B) Left valve view of the cell showing the absence of pores at the pyrenoid region (C) Right valve view of the cell showing the absence of pores at the pyrenoid region (0) Side view of the cell Arrow showing marginal pores along the circumference of the cell (E) Prominent notch with the eight plates labeled accordingly (F) The apical pore region where the flagellar pore f is larger than the auxiliary pore a

310 Cells ofProrocentrum rhathymum in culture tube showing planktonic 126 cells

Xlll

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Figure Page 212 Scanning electron micrographs of Diplosalis lenticula from Tebrau 49

Strait Johor (A) Apical view showing the apical pore (ap) (B) Antapical view (C) Dorsal view with the sulcus

2l3 Scanning electron micrographs of Diplopsalopsis orbicularis covered 50 in dirt Scale bar = 10 )tm

214 Scanning electron micrographs of Gonyaulax cf scrippsae from 51 Cherating Pahang (A) Lateral view of cell with large trichocyst pores (B) Anterio-ventral view of cell showing two cingulum displacement and sulcus (s)

215 Scanning electron micrograph of Gonyaulax polygramma from 53 Cherating Pahang

216 Light micrographs of Gonyaulax spinifera from Semariang Sarawak 54 (A) Cell with apical horn (arrow) (B) Red autofluorescence showing chloroplast content Scale bar = 10 )tm

217 Scanning electron micrographs of Gymnodinium instriatum from 56 Santubong Sarawak (A) Ventral view showing the cingulum displacement (B) Dorsal view showing slightly bilobed hypocone and conical epicone (C amp D) Antapical view showing the sulcal intrusion into the antapex Scale bar = 5 )tm

218 Scanning electron micrograph of Gyrodinium spirale from Tebrau 58 Strait Johor Scale bar = 20 )tm

219 Scanning electron micrograph of Gymnodinium mikimotoi from 60 Tebrau Strait Johor showing vertical apical groove (ag)

220 Scanning electron micrographs of Karlodinium veneficum from 63 Tebrau Strait Johor (A) Apical-dorsal view showing elongated ventral pore (vp) and apical groove (ag) (B) Dorsal view showing clearly the 3x cingulum displacement (cd) descending to the right (C) Ventral view showing the starting of apical groove Scale bar = 5 )tm

221 Scanning electron micrographs of Lingulodinium polyedrum from 66 Clterating Pahang

222 Light and scanning electron micrographs of Neocertium furca from 68 Tebrau Strait Johor (A) SEM showing the cell and the longitudinal ridges (B-C) LM Cells from Semariang Sarawak (B) A chain of two vegetative cells (C) Red autofluorescence showing chloroplast content Scale bar = 20 )tm

223 Light micrographs of Noctiluca scintillans from Santubong Sarawak 69 (A) Cells with greenish spots (B) Apical view (C) Antapical view showing flagellum

224 Light and scanning electron micrographs of Prorocentrum gracile 71 from Semariang Sarawak (A-B) LM (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-D) SEM Scale bar = 10 )tm

XI

Figure Page 225 Scanning electron micrograph of Prorocentrum micans from Tebrau 73

Strait lohor

226 (A) Light micrograph of Prorocentrum rhathymum showing short 74 apical spine (arrow head) (B) Red auto florescence showing chloroplast content (C-F) Epiflorescence micrograph (C) Right valve (D) Left valve (E) Periflagellar area (arrow head) (F)

Intercalary band (arrow) Scale bar = 10 pm

227 Scanning electron micrographs of Protoceratium reticulatum from 76 Cherating Pahang (A) Lateral view Cingulum displacement (arrow) (B) Dorsal-antapical view

228 Scanning electron micrograph of a new morphotype Protoceratium 78 sp I from Cherating Pahang showing the ventral-antapical view

229 Scanning electron micrograph of Protoperidinium latissimum from 79 Cherating Pahang Ventral view

230 Scanning electron micrographs of Protoperidinium marukawai from 80 Cherating Pahang Scale bar = 10 urn

231 Scanning electron micrographs of Protoperidinium pyriforme from 81 Cherating Pahang (A) Dorso-antapical view showing two antapical

spines (B) Dorsal view Apical horn is present (arrowhead) Scale bar = 10 pm

232 Scanning electron micrograph of Peridinium quinquecorne from 82 Cherating Pahang Ventral view

233 Scanning electron micrograph of Protoperidinium sp I from 83 Cherating Pahang Apical ventral view

234 Scanning electron micrograph of Protoperidinium sp 2 from 84 Kampung Geliga Besar Terengganu Dorsal view One of the antapical spine is covered in dirt

235 Scanning electron micrograph of Protoperidinium subinerme from 85 Cherating Pahang Apical view

236 Light micrograph of Pyrodinium bahamense var compressum from 86 Port Dickson Negeri Sembilan

237 Light micrographs of Pyrophacus stein from Semariang Sarawak 88 (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-H) Calcoflour stained ceUs showing thecal plates

238 Scanning electron micrograph of Scrippsiella trochoidea from Tebrau 89 Strait Johor Scale bar = 10 pm

XII

Figure Page 31 Clonal culture of Coolia malayensis showing the string of mucilage 100

(arrow) formed at the surface of the medium

32 Scanning electron microscopy of Coolia malayensis from Lundu 103 Sarawak (A) Apical view showing the large 6 (B) Dorsal view showing apical pore Po (C) Apical-ventral view showing I and 7 plates (0) Ventral view showing sulcus (E-F) Antapical-ventral views showing the large 3 (G) Apical pore complex showing long silt of apical pore (H) Minute perforations were observed within the surface pores (I) Lateral view of the cell

33 Phylogenetic tree of Coolia species derived from Bayesian analysis 107 of LSU rONA sequences The C malayensis strains from Lundu Sarawak used in this study are in boldface MP and ML bootstrap and BI posterior probability values are shown at the left of the internal nodes (MPIMLIBI) The four end-clades were labeled as Clade I 11 III and IV for comparison Scale bar =01 substitution per site

34 Secondary structural diagram of Coolia malayensis (CmLDO I) ITS2 109 transcript from Lundu Sarawak with universal motif shown AAA and GUU motif showing sequence conservation for 14 ITS2 sequences analyzed Arrow indicates U-U mismatch

35 Minimum spanning network of ITS haplotypes found in Malaysian 112 Coolia malayensis populations

36 Profile-neighbor joining (PNJ) tree of Coolia species Lundu strains 114 of C malayensis are in boldface

37 Gambierdiscus belizeanus GdSA03 from Kota Kinabalu Sabah (A) 119 LM Lugols stained cell showing the short longitudinal flagellum (arrow) (B) Epifluorescent micrographs of divided cells showing both epi- and hypotheca (C) Ventral view of cell showing first precingular plate (I ) and trapezoid 7 immersed into the ascending cingUlum and the deep sulcus Note the striation of the intercalary bands and the ridged margin of cingulum list (0) Apical view of cel l (E-F) antapical view of cell I p is small and narrow (G) Close view of the apical pore complex (APC) (H) Close view of the sulcal region showing the Sda t Sdp Ssp and Ssa plates

38 Prorocentrum lima strain PLKdO 1 from Kudat Sabah (A) Culture in 122 flask Clump of cells floating in the water column due to bubbles produced during photosynthesis activity (B) P lima cell observed under LM with 200x magnification Pyrenoid is located in the center of the cell (arrow)

39 Prorocentrum lima from Kudat Sabah SEM (A) valve views 125 showing two cells with left and right valves (B) Left valve view of the cell showing the absence of pores at the pyrenoid region (C) Right valve view of the cell showing the absence of pores at the pyrenoid region (0) Side view of the cell Arrow showing marginal pores along the circumference of the cell (E) Prominent notch with the eight plates labeled accordingly (F) The apical pore region where the flagellar pore f is larger than the auxiliary pore a

310 Cells ofProrocentrum rhathymum in culture tube showing planktonic 126 cells

Xlll

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Figure Page 225 Scanning electron micrograph of Prorocentrum micans from Tebrau 73

Strait lohor

226 (A) Light micrograph of Prorocentrum rhathymum showing short 74 apical spine (arrow head) (B) Red auto florescence showing chloroplast content (C-F) Epiflorescence micrograph (C) Right valve (D) Left valve (E) Periflagellar area (arrow head) (F)

Intercalary band (arrow) Scale bar = 10 pm

227 Scanning electron micrographs of Protoceratium reticulatum from 76 Cherating Pahang (A) Lateral view Cingulum displacement (arrow) (B) Dorsal-antapical view

228 Scanning electron micrograph of a new morphotype Protoceratium 78 sp I from Cherating Pahang showing the ventral-antapical view

229 Scanning electron micrograph of Protoperidinium latissimum from 79 Cherating Pahang Ventral view

230 Scanning electron micrographs of Protoperidinium marukawai from 80 Cherating Pahang Scale bar = 10 urn

231 Scanning electron micrographs of Protoperidinium pyriforme from 81 Cherating Pahang (A) Dorso-antapical view showing two antapical

spines (B) Dorsal view Apical horn is present (arrowhead) Scale bar = 10 pm

232 Scanning electron micrograph of Peridinium quinquecorne from 82 Cherating Pahang Ventral view

233 Scanning electron micrograph of Protoperidinium sp I from 83 Cherating Pahang Apical ventral view

234 Scanning electron micrograph of Protoperidinium sp 2 from 84 Kampung Geliga Besar Terengganu Dorsal view One of the antapical spine is covered in dirt

235 Scanning electron micrograph of Protoperidinium subinerme from 85 Cherating Pahang Apical view

236 Light micrograph of Pyrodinium bahamense var compressum from 86 Port Dickson Negeri Sembilan

237 Light micrographs of Pyrophacus stein from Semariang Sarawak 88 (A) Vegetative cell (B) Red autofluorescence showing chloroplast content (C-H) Calcoflour stained ceUs showing thecal plates

238 Scanning electron micrograph of Scrippsiella trochoidea from Tebrau 89 Strait Johor Scale bar = 10 pm

XII

Figure Page 31 Clonal culture of Coolia malayensis showing the string of mucilage 100

(arrow) formed at the surface of the medium

32 Scanning electron microscopy of Coolia malayensis from Lundu 103 Sarawak (A) Apical view showing the large 6 (B) Dorsal view showing apical pore Po (C) Apical-ventral view showing I and 7 plates (0) Ventral view showing sulcus (E-F) Antapical-ventral views showing the large 3 (G) Apical pore complex showing long silt of apical pore (H) Minute perforations were observed within the surface pores (I) Lateral view of the cell

33 Phylogenetic tree of Coolia species derived from Bayesian analysis 107 of LSU rONA sequences The C malayensis strains from Lundu Sarawak used in this study are in boldface MP and ML bootstrap and BI posterior probability values are shown at the left of the internal nodes (MPIMLIBI) The four end-clades were labeled as Clade I 11 III and IV for comparison Scale bar =01 substitution per site

34 Secondary structural diagram of Coolia malayensis (CmLDO I) ITS2 109 transcript from Lundu Sarawak with universal motif shown AAA and GUU motif showing sequence conservation for 14 ITS2 sequences analyzed Arrow indicates U-U mismatch

35 Minimum spanning network of ITS haplotypes found in Malaysian 112 Coolia malayensis populations

36 Profile-neighbor joining (PNJ) tree of Coolia species Lundu strains 114 of C malayensis are in boldface

37 Gambierdiscus belizeanus GdSA03 from Kota Kinabalu Sabah (A) 119 LM Lugols stained cell showing the short longitudinal flagellum (arrow) (B) Epifluorescent micrographs of divided cells showing both epi- and hypotheca (C) Ventral view of cell showing first precingular plate (I ) and trapezoid 7 immersed into the ascending cingUlum and the deep sulcus Note the striation of the intercalary bands and the ridged margin of cingulum list (0) Apical view of cel l (E-F) antapical view of cell I p is small and narrow (G) Close view of the apical pore complex (APC) (H) Close view of the sulcal region showing the Sda t Sdp Ssp and Ssa plates

38 Prorocentrum lima strain PLKdO 1 from Kudat Sabah (A) Culture in 122 flask Clump of cells floating in the water column due to bubbles produced during photosynthesis activity (B) P lima cell observed under LM with 200x magnification Pyrenoid is located in the center of the cell (arrow)

39 Prorocentrum lima from Kudat Sabah SEM (A) valve views 125 showing two cells with left and right valves (B) Left valve view of the cell showing the absence of pores at the pyrenoid region (C) Right valve view of the cell showing the absence of pores at the pyrenoid region (0) Side view of the cell Arrow showing marginal pores along the circumference of the cell (E) Prominent notch with the eight plates labeled accordingly (F) The apical pore region where the flagellar pore f is larger than the auxiliary pore a

310 Cells ofProrocentrum rhathymum in culture tube showing planktonic 126 cells

Xlll

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Figure Page 31 Clonal culture of Coolia malayensis showing the string of mucilage 100

(arrow) formed at the surface of the medium

32 Scanning electron microscopy of Coolia malayensis from Lundu 103 Sarawak (A) Apical view showing the large 6 (B) Dorsal view showing apical pore Po (C) Apical-ventral view showing I and 7 plates (0) Ventral view showing sulcus (E-F) Antapical-ventral views showing the large 3 (G) Apical pore complex showing long silt of apical pore (H) Minute perforations were observed within the surface pores (I) Lateral view of the cell

33 Phylogenetic tree of Coolia species derived from Bayesian analysis 107 of LSU rONA sequences The C malayensis strains from Lundu Sarawak used in this study are in boldface MP and ML bootstrap and BI posterior probability values are shown at the left of the internal nodes (MPIMLIBI) The four end-clades were labeled as Clade I 11 III and IV for comparison Scale bar =01 substitution per site

34 Secondary structural diagram of Coolia malayensis (CmLDO I) ITS2 109 transcript from Lundu Sarawak with universal motif shown AAA and GUU motif showing sequence conservation for 14 ITS2 sequences analyzed Arrow indicates U-U mismatch

35 Minimum spanning network of ITS haplotypes found in Malaysian 112 Coolia malayensis populations

36 Profile-neighbor joining (PNJ) tree of Coolia species Lundu strains 114 of C malayensis are in boldface

37 Gambierdiscus belizeanus GdSA03 from Kota Kinabalu Sabah (A) 119 LM Lugols stained cell showing the short longitudinal flagellum (arrow) (B) Epifluorescent micrographs of divided cells showing both epi- and hypotheca (C) Ventral view of cell showing first precingular plate (I ) and trapezoid 7 immersed into the ascending cingUlum and the deep sulcus Note the striation of the intercalary bands and the ridged margin of cingulum list (0) Apical view of cel l (E-F) antapical view of cell I p is small and narrow (G) Close view of the apical pore complex (APC) (H) Close view of the sulcal region showing the Sda t Sdp Ssp and Ssa plates

38 Prorocentrum lima strain PLKdO 1 from Kudat Sabah (A) Culture in 122 flask Clump of cells floating in the water column due to bubbles produced during photosynthesis activity (B) P lima cell observed under LM with 200x magnification Pyrenoid is located in the center of the cell (arrow)

39 Prorocentrum lima from Kudat Sabah SEM (A) valve views 125 showing two cells with left and right valves (B) Left valve view of the cell showing the absence of pores at the pyrenoid region (C) Right valve view of the cell showing the absence of pores at the pyrenoid region (0) Side view of the cell Arrow showing marginal pores along the circumference of the cell (E) Prominent notch with the eight plates labeled accordingly (F) The apical pore region where the flagellar pore f is larger than the auxiliary pore a

310 Cells ofProrocentrum rhathymum in culture tube showing planktonic 126 cells

Xlll

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Figure Page 311 Prorocentrum rhathymum from Semariang Sarawak LM (A) Cell 127

with light brown chloroplast (B) Red autofluorescence showing the

chloroplast content in the cell

312 Prorocentrum rhathymum from Semariang Sarawak SEM (A) Left 128 valve view showing rows trichocyst pores perpendicular with the

intercalary band (B) Right valve view with flagellum (arrow head)

(C) Arrow showing narrow intercalary band of new cells (D) Arrow

showing thicken intercalary band from dividing cell (E) Arrow

showing flagellum from the peri flagellar area (F) Arrow showing

platelets in the peri flagellar area

313 Phylogentic inference of Prorocentrum species derived from ML 132 analysis ofLSU rONA sequences P rhathymum strain from Semariang Sarawak is in boldface The MPML bootstrap and BI posterior probability values are shown at the left of the internal nodes Scale bar =01 substitution per site

314 Protoceratiumfukuyoii sp nov from Semariang Sarawak LM (A) 134 Cells with golden-brown chloroplasts (B) Red autofluorescence

showing chloroplast content (C) epi-fluorescence image of cell

stained with Calcofluor White showing the ventral view of cell

315 Protoceratiumfukuyoii sp nov from Semariang Sarawak SEM (A) 136 Ventral view showing the sa (anterior sulcal plate) precingular plates 1 5 and 6 (B) Apical view showing apical plates 1 - 3 anterior intercalary plate 1 a and precingular plates 1- 6 (C) Close up of apical pore complex showing a slit-like Po with a few marginal pores Scale bar = 2 lm (D) Lateral antapical view showing post cingular plates 1 - 3 posterior intercalary plate 1 p and antapical plate 1 (E) Anterior view showing post cingular plates 3 - 6 and antapical plate 1 (F) Lateral view showing the slightly reticulated epicone and heavy reticulated hypocone Scale bar = 5lm

316 Secondary structural diagram of ITS2 transcript from Protoceratium 137 fukuyoii sp nov (GgSmO 1) with universal motif shown AAA and UGG motif showing sequence conservation for 6 ITS2 sequences

analyzed Arrow indicates U-U mismatch

317 Comparison of secondary structures of Protoceratium fukuyoii sp 138 nav (GgSmOl) ITS2 transcript from Semariang and P reticulatum (EU927567) revealed 2 CBCs and 2 hemi-CBCs in Helix I 3 hemi-

CBCs in Helix III and 1 hemi-CBC in Helix IV

318 Bayesian tree inferred from LSU region of rONA of Protoceratium 140 and Gonyaulax species with Akashiwo sanguinea as outgroup -In = 199633788

319 Culture ofAlexandrium tamiyavanichii in culture tube showing 141 planktonic cells

320 Alexandrium tamiyavanichii strain AcSmO I from Semariang 142 Sarawak LM (A) A chain of two vegetative cells (B) Red

autofluorescence showing the chloroplast content Scale bar == 10 lm

XIV

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Figure Page 321 Alexandrium tamiyavanichii (strain AcSmO I) from Semariang 143

Sarawak Epifluorescence micrographs (A) Chain forming of four vegetative cells (B) Apical-ventral view of cell prp precingular part sa anterior sulcal plate Theres an oblique posterior end of 1 and a triangular shape of the ppr Apical plates 1- 4 and precingular plates 1 2 4 - 6 (C) Dorsal - apical view showing apical pore (Po) and precingular plates 2 - 5 (D) Close-up of the Po (E) Apical view showing the ventral pore (vp) (F) Antapical-ventral view showing postcingular plates I 4 5 and antapical plate 1 (G) Dorsal-antapical view showing postcingular plates 2 34 and antapical plate 2 Scale bars = 10 Ilm

322 HPLC chromatogram of A tamiyavanichii strain AcSMO 1 from 144 Samariang (A) Toxin standard (B) Crude extract of A

tamiyavanichii AcSMOI

323 Sequence logo of the A tamiyavanichii signature 145 324 Secondary structural diagram ofAlexandrium tamiyavanichii 146

(AcSmOl) ITS2 transcript from Semariang Sarawak with universal AAA and UGGU motif showing sequence conservation for 5 ITS2 sequences analyzed Arrow indicates U-U mismatch

325 Secondary structural diagram ofAlexandrium tamiyavanichii 147 (A 1PSA) ITS2 transcript from Brazil with 3 Hemi-CBCs from Helix I II and III respectively A base change and insertion at the Helix IV

326 Phylogenetic tree ofAlexandrium tamiyavanichii inferred from BI of 149 ITS sequences A tamiyavanichii strain from Semariang Sarawak used in this study is in boldface MP and ML bootstraps and BI posterior probability values are shown at the internal nodes Scale bar =01 substitution per site

xv

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

CHAPTER I

INTRODUCTION

11 DinoOagellates and red tides

Algal bloom are generally known to the public as red tide The observed reddish patches

of seawater are originated from high concentrations of dinoflagellates photopigment

peridinin A wide range of research has been conducted on this group of phytoplankton due

to the unusual blooming phenomenon that happens occasionally in the water Algal blooms

usually refer to the abnormal growth of a certain species of phytoplankton in the water

column (exceeding 106 cells L- 1) in a short period of time at favorable conditions (Us up et

al 1989) Although there are green tide and brown tide due to different species of algal

blooms the public generally call this kind of toxic bloom as red tide Since these algal

bloom produces toxin and causes physical damage to human and other life forms and the

tenn hannful algal blooms (HABs) was coined HABs are often related to shellfish and

fish poisonings Filter-feeding shellfish mollusks that ingest large quantity of toxic

phytoplankton are served as the vectors in which the toxin is transferred to the higher

trophic level via food chain Accumulation of toxins in the shellfish may not give any

negative effects to the shellfish but the toxins may cause fatality to mammals birds and

fishes These included the commercially available bivalves such as green mussels (Perna

viridis) razor clam (Solen sp) carpet clam (Paphia undulate) cockles (Anadara

granosa) benthic clam (Polymesoda sp) and sea snails (Oliva sp) (Smith 2001)

Contaminated shellfish with different type of toxins lead to different form of shellfish

poisonings which included paralytic shellfish poisoning (PSP) amnesic shellfish

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

poisoning (ASP) diarrheic shellfish poisoning (DSP) neurotoxic shellfish poisoning

(NSP) and azaspiracid shellfish poisoning (AZP) Reef fishes like parrot fish are also one

ofthe toxin vectors (Jothy 1984) which lead to ciguatera fish poisoning (CFP)

High phytoplankton density may also clog fish gills causing fish to suffocate and

die When high density of phytoplankton blooms die off and decompose in the natural

waters it creates an anoxia environment where other living organisms die due to

insufficient oxygen level This usually causes massive fish kills in the enclosed aquaculture

fanns (Glibert et aI 2002 Kempton et aI 2002 Whyte et aI 200 l )

12 History of HABs in Malaysia

The main HABs causative organisms in Malaysia are Pyrodinium bahamense var

compressum Alexandrium minutum and A tamiyavanichii (Lim et aI 2004 Usup et aI

2002b) They are the PSP causative species that produced a potent neurotoxin saxitoxin

(STX) This group of toxin blocks sodium channels on the nerve cells and smooth muscles

resulting in heart failure disrupting respiratory system and eventually causes death

(Shimizu et aI 1985) In humans victims will experience a tingling burning down the

throat and graduaUy extend to the extremities The body will then feel numb and unable to

make any movement Finally there will be paralysis of upper and lower limbs and extreme

respiratory distress which might cause death if toxin levels are high (Halstead and Schantz

1984)

Previous studies also showed the existence of other HABs species such as

Dinophysis spp Prorocentrum lima P micans Gambierdiscus toxicus Ostreopsis spp

Coolia spp Cochlodinium spp and Pseudo-nitzschia spp in Malaysian waters (Anton et

at 2008 Leaw et aI 2010 Leaw et aI 2001 Lewis and Holmes 1993 Lim et aI 2004)

Some of these toxic species cause DSP CFP ASP and fish kills

2

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Malaysia is the first country in the Southeast Asia region that faces the problem of

P bahamense var compressum The first outbreak in January to May 1976 caught

Malaysian in surprised when around 300 km of the west coast of Sabah was affected and

202 PSP cases including seven deaths were reported (Roy 1977) According to the

statistics from Fisheries Department of Sabah there were 325 cases of PSP including 31

fitalities between the years 1976 and 1988 (Ting and Wong 1989) Information compiled

showed that fatality cases were caused by consuming contaminated clams oysters giant

clam cockles and mussels (Ting and Wong 1989) In Sabah shellfish ban is a usual

routine early of each year From year 2004 to 20 I 0 HAB was detected along the west

coast of Sabah (Usup et aI 20 II) Blooms normally started at the coastal area of Kota

Kinabalu including Gaya Island Sepanggar Bay and Kuala Penyu and spread southward

to the Brunei Bay and northward to Kudat Although toxicity level of some blooms

reached 1000 mouse units (MU) shellfish ban had minimized the fatality to one case in

Menggatal and reduced hospitalized cases throughout the blooming period

In late January 2007 Cochlodinium polykrikoides bloom was reported in Sabah

which killed the cultured groupers in Gaya Island (Anton et aI 2008) Gymnodinium

catenatum was also reported from the same area at the same time (Adam et aI 2011)

Various precautions methods were introduced by the Fisheries Department of

Sabah after the PSP incident including monitoring the toxin levels in shellfish and plankton

fortnightly-sampling and monitoring In Sabah when the toxin in shellfish mollusks is

exceeding the limit of 80 IlgiIOO g a public warning will be issued via local radio

broadcast directly to ensure the public can receive the information in the shortest time

Those who ignore the warning are most likely to be the next PSP victims (Ting and Wong

1989) Although bloom of P bahamense occur almost annually in the state of Sabah PSP

3

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

events due to P bahamense had greatly decreased through the efficient shellfish toxicity

monitoring program and increased public awareness (Usup and Azanza 1998)

Until 1990 HAB related problems were always confined to the state of Sabah

However in early 1991 three persons were reported ill after consuming green mussels

(Perna viridis) harvested from a recent established aquaculture farm in Sebatu Malacca

This was the first reported PSP case in the Peninsular Malaysia (Usup et aI 2002c)

Surprisingly ten potential HAB species were identified but P bahamense was not in the

list Two potentially PSP toxin producers Alexandrium tamiyavanichii and Gymnodinium

catenatum were suspected to be the causative organisms in that incident due to the high

toxin level which reached 325 MU (Anton et aI 2000)

Ten years later another PSP case was reported from the east coast of Peninsular

Malaysia (Lim et aI 2004) In September 2001 six people was hospitalized including one

fatality was reported from Tumpat Kelantan Victims suffered PSP symptoms after

consuming benthic clams ( lokan ) Polymesoda sp collected from a lagoon located at

Sungai Geting Tumpat That was the first report of toxic dinoflagellate Alexandrium

minutum in Malaysian waters (Lim et aI 2004) The blooming event spread to the

southern part of Peninsular Malaysia in 2002 The Johor Department of Environment

(DOE) spotted reddish water at Lido Beach in 1i h July 2002 (New Straits Times 2002)

However the causative organism was later identified as Prorocentrum minimum

(Rozirwan 2010)

At the north-western of Peninsular Malaysia the Penang Department of

Environment (DOE) reported the occurrence of red tides in Pulau Aman Teluk Bahang

Batu Maung Kuala Muda and Kuala Juru in July 2007 Non-toxic but harmful

Neoceratium Jurea was identified as the causative organism of the bloom However the

quantity was not dense enough to choke up the gills offish and no fish kill was reported

4

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

PUllt Kbidmat Maldumat Akademik UNIVERSm MALA SIA SARAWAJ(

In 2010 fish kills was observed by fishermen at Gelang Patah Johor Witness

testified that the water turned yellowish and murky and they could smell a terrible odor

from the site Dead fish can be seen floating along a 5 km stretch on the sea The bloom

affected 50 fish fanns off Pulau Ubin with estimated loses of RM 800000 per farm The

cultured fish included the tiger grouper sea bass and red snapper (Daily Express 2010)

5

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

Table tt Historical event ofHABs in Malaysia ftom 1916 - 2010

Year Causative organism Location Impact References

1976 Pyrodinium bahamense var

compressum

J991 Aletandrium tamiavanichi and

Gymnodinium catenatum

200 I Alexandrium minutum

2002 Prorocentrum minimum

2005 Cochlodinium polykrikoides

2006 Cochlodinium polykrikoides

2007 Cochlodinium polykrikoides

2007 Neoceratium furca

2009 Pyrodinium bahamense

2010 Unknown

Sabah

Sebatu Malacca

Tumpat Kelantan

Johor Bharu Johor

Prai Penang

Kuching Sarawak Kota

Kinabalu Sarawak

Pulau Gaya Sabah

Pangkor Lumut Penang

Kota Kinabalu and

surrounding areas

Gelang Patah Johor

202 PSP cases seven fatalities

Three il l after consuming green

mussels (Perna viridis)

Six were hospitalized including one

casualty

Water discoloration

Water discoloration and Massive fish

kill at aquaculture cage

Water discoloration some fish kills

Cultured groupers died

Water discoloration

Shellfish contamination gt7000 MU

ban of shellfish harvesting

Fish kills affected 50 farms estimate

loss of RM 800000

Roy 1977 Fisheries

Department Sabah

Anton et aI 2000

Lim et aI 2004

Adam et aI 20 II

Fisheries Research Institute

Bintawa Sarawak

Anton et aI 2008 Daily

Express (2007)

Fisheries Research Institute

Batu Maung Penang

Fisheries Department Sabah

Express Daily (2009)

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

13 Paralytic Shellfish Poisoning (PSP)

Malaysia is one of the countries that are affected by the food borne disease paralytic

shellfish poisoning (PSP) This type of toxin intoxication results from the consuming of

shellfish mollusks containing the potent neurotoxins accumulated from toxic

dinoflagellates

PSP is caused by the ingestion of filter-feeding shellfish contaminated with

saxitoxin (STX) which block mammalian sodium channels in the nervous system

preventing the transmission of neuron signal and thus cause mild symptoms such as

paralysis and death in more severe cases Phytoplanktons associated with PSP are members

from the genera Pyrodinium Alexandrium and Gymnodinium PSP is one of the major

public health risks in the Southeast Asian region (Usup et ai 2011 Usup and Azanza

1998)

131 The causative organisms

Pyrodinium bahamense

Pyrodinium bahamense is the most important paralytic shellfish toxin (PST) producing

dinoflagellates in tropical waters In 1976 the species was reported in Malaysia for the first

time Pyrodinium is now accepted as a monospecies with two varieties namely var

bahamense and var compressum Various studies had been done on this species from cell

isolation and culturing techniques (Azanza-Corrales and Hall 1993) encystment of the

cells (Corrales et ai 1995) cyst density (Corrales and Crisostomo 1996) cyst distribution

(Furio et aI 1996) cells transportation by water currents (Sotto and Young 1995) growth

and toxin production (Usup et ai 1994) saxitoxins and related toxins in the shellfish

(Harada et aI 1982Usup et ai 1995) as well as phylogenetic analysis (Leaw et ai 2005)

7

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8

11 Scanning micrographs of Pyrodinium bahamense var compressum from Kota Kinabalu Sabah (source Lim 1999 Leaw et aI 2005)

Mmlwldrium species

ADJOII12 the taxonomically accepted 30 species of Alexandrium 13 species from the genus

known to produce STX and results in PSP intoxication The harmful species are

atrDJldrium acatenela A andersonii A catenella A cohorticula A fundyense A

i A leei A minutum A monilatum A ostenjeldii A pseudogonyaulax A

teristics for taxonomical observation (Balech 1995) ultrastructures (Phanichyakarn

at 1993) technique to identify Alexandrium cysts in the environment (Yamaguchi et

1995) germination of the cysts from sediments (Cannon 1993) bloom patterns and

ity of the shellfish in different areas (Sekiguchi et ai 1996) detection of toxin in

Ufish fed with cultured Alexandrium (Wisessang et ai 1991) toxin composition of

under different parameters in culture or environment (Anderson et ai 1990 Flynn

8