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EVOLUTIONARY LINEAGE OF NAKED HARMFUL , KARLODINIUMIKARENIAITAKA YAMAI GYRODINIUM COMPLEX ()

Chow Luan Jia

Bachelor of Science with Honours (Resource Biotechnology) 2011 ~ ..sa. u...... , i.dl...... 'AUl1fllu lNWM1I M&LArM SAMWAt..

EVOLUTIONARY LINEAGE OF NAKED HARMF L DU OFLAGELLATES KARLODINIUMI KAREN/AI TAKA yAft:L!V GYRODINIUM COMPLEX mINOPIlYCEAE)

Chow Luan .Jia (20813)

This project is submitted in partial fullfilment of the requirements of the degree of Bachelor of Science with Honours (Resource Biotechnology)

upervisor: Dr Leaw Chui Pin

Co-supervisor: Dr Lim Po Teen

Resource Bioctchnolog} Prngrfl lnme Depanmenl of Molecular Biolog} Faeulty of Resource Science and Tt:c: hnology lIni\cr!'oIlY Malaysia Sanl\\ak

2011

"·t • • • DECLARATION

I hereby declare that no portion of the \ ork referred to this thesis has been submitted in

support of an appl ication for another degree of qualification to this or any other univ rsity

nr institution of higher Ieaming.

(~~ CHOW LUA, JIA

Resource Biotechnology Programme,

Faculty of Resource Science and Technology,

University Malaysia Sarawak.

, ...... ill -. . A KNOWLEDGEMENTS

First fall, [ would like to lhank Universiti Malaysia Sarawak for giving me this opportunity to complete my fmal year rojecl. The greatest honors go to my supervisor Dr

Leaw Chui Pin and CO-5upervisor Dr Lim Po Teen for their leadership and guidance in completing the study. Sin erely thank.> to the Sarawak Fisheries Department for the acccssibility to the sampling site.

Great appreciation to the following individuals for their as istances In varIous forms: Hii Kien Soon, Tan Toh Hii, Lim Hong Chang, Teng Sing Tung and all the lab members and the lab assistants of the ECOlOxicology Laboratoy and !BEC Molecular

Laboratory. My gratitudes also go to the FRST science officers especiall y En. SafTi, En.

Besar_ En. Nazri, and Mdm. Ting Woei for their helps and hospitality.

Last but not least, I would like to thank my family for thei r financial, moral and emotional supports. My siblings receive my deepest gratitude for their dedication and support during my undergraduate studies that provided the foundati on for this study.

This project was supported by MOSTI eScience Fund to Dr Leaw. TABLE OF CONTENTS

Page ACKNOWLEDGEME TS TABLE OF CO:'olTENT. ii LI T OF ABBREVIATIONS iv

LI T OF FIGURE \

LIST OF TABLES Vll ABSTRACT viii ABSTRAK viii

1.0 L"ITROD CTION

2.0 LITERATURE REVIEW 3 2.1 Naked dinoflagellates 3 22 Harmful alga blooms 7 2.3 History of neurotoxic shellfish poisoning (NS?) 8 2.4 Ribosomal RNA genes (rONA) region 10

3.0 MATERIA LS AND METHODS I I 3.1 Sample ollection and clonal culru rc c'lablishment II 3.2 Species identification 12 3.3 Genomic 0 A extraction J3 3.4 Amplification and sequencing of rONA 13 3.5 Phylogenetic analyses 14 3.5. 1 Sequence analysis and taxon ,. ampling 14 3.5.2 LSU phylogenetic analysis 15 3.5.3 Matrix construction for morphological ·:·:laraclers J5

11

. ------­ ~. 4.0 RESULTS 16 4.1 Algal cul tures establ i hed 16 4.2 Species identification 16 4.1.1 Protocerarium rericuiatum 17 4.2 .2 Prorocentrum rhathymum 19 4.2.3 GyrodmiulII illslriarum 21

')' 4.2.4 Alexandrilllll sp. - ~ 4. 2.5 Akashill 0 sal1guinea 24 4.2.6 ochlodinium cf. p ofykrikoides 25 4.2. 7 Prorocentrum sigmoides 26 4.2.8 Karlodinium veneficwn 27 4.3 Genomic DNA extraction, amplification and purification 29 4.4 Taxon sampling 30 4.5 Phylogenetic inferences of naked dinoflagell ates 31 4.5.1 Karlodinium phylogeny 31 4.5.2 phylogeny 32 4.5 .3 Takayama phylogeny 33 4.5.4 Gyrodinium phylogeny 34 4.6 Morphological traits 35 4.7 Matrices constructed for character state evolution 47 4.8 Character state evolution 50 4.8.1 Character state evoluti on of Karlodiniwn 50 4.8.2 Character state evolution of Karenia 53 4. 8.3 Character state evolution of Taka.vama 56 4.8.4 Character state evolution of Gyrodinillm 58

5.0 DISCUSSION 60

6.0 CO CLU ION 69

7.0 REFERENCES 70

III

.; ------~, LIST OF ABBREVI TJONS

N P Neurotoxic Shellfi sh Poisoning

BLAST Basic local aJigIunent search tool

EM Scanning Electron Microscope

LM Light Microscope

HAB Harmful algal bloom

rRNA Ribosomal genes

CPO Cri tical Point Dried

IV

... ---- ~, Ll T OF FIGURE

P ge Figure 2.1 Phylogeny of major g nera of dinoflag Ha tes. trict consensus 6 of the 10 equally parsimonious trees obtained with the heuristic 'eareh option in P UT' (Oaug~i erg . ~OOO).

FIgur· ~ _. , ')_ The A.arenia brevis, the causative organism of red 10 tides on tb West Florida sbelf (Da\,id Partersoo, 1arille Biological Laboratory cited in Lorraine, 2006).

Figure 3.1 Map :ho\\ing 'antubong and Semariang sampling site. 13

Figure -t .1 Light and scanning electron micrographs of Pr%eem/illn! 18 reticularum fro m Semariang, Sarawak.

Figure 4.2 Light and scaMing electron micrographs of Prorocenrrum 20 rha/hymum from Semariang, Sarawak.

Figure 4.3 Light micrographs of inslriarum from Santubong, 21 Sarawak.

Figure 4.4 ScaMing electron micrographs of Gymnodinium ins/ria/urn from 22 Saotubong, Sarawak.

Figure 4.5 Light and scanning electron micrographs of Alexandrium sp. 23 from Semariaog, Sarawak.

Figure 4.6 Light and autofluorescence micrographs of Akashiwo sangllinea 24 from Semariang, Samwak.

Figure 4.7 Light aod autofluorescence micrographs of Cochlodiniwn cf. 25 polykrikoides from Semariang, Sarawak.

Figure 4.8 Light and autofluorescence micrographs of Prorocentrum 26 sigmoides fro m Selllarian g, Sarawak.

Figure 4.9 Scanning electron micrographs of Karlodinium venejiculIl from 28 Johore.

Figure 4.10 Negative image of gel for purified PCR products of LS rONA 29 gene amplified from dinoflagellate cultures. L, 1000bp ladder (Promega, U A); lane I, GiSB30; lane 2, Al SM86; lane 3, AISM94; lane 4, CoLD I 0 and -ve, negative control.

Figure 4. 12 MP tree of Kariodinilllll with tree length 01' 61 2 evolutionary 31 steps and bootstrap \'alue of 1000. The consistenc) index (el) was 0.8284 and retention index IRJ ) - 0.6602.

v

.; ------~; Figure 4.12 MP tree of Karenia with tree length of 480 evolutionary steps 32 and bootstrap value of 1000. The consistency index (CI) was 0.8583 and retention index (Rl) =0.5613.

Figure 4.13 MP tree of Takayama with tree length of 392 e olutionary steps 33 and bootstrap yulue of \000. The consistency index (CI) was 0.9337 and retention index (RI) =0.7204.

Figure 4.14 !'vIP tree of Gyrodinillm with tree length of 745 evolutionary 34 steps and bootstrap value of 1000. n1e consistency index (Cl ) was 0.8389 and retention index tID) =0.53-19.

Figure ·U 5 Character states mapping onto the MP tree of genus 52 Karlodinillln with 15 character states and I I taxa including 3 outgroups.

Fi gure 4.16 Character states mapping onto the MP tree of genus Karenia 55 with 17 character states and 9 taxa including 3 outgroups.

Figure 4.17 Character states mapping onto the MP tree of genus Takayama 57 with 15 character states and 8 taxa including 3 outgroups.

Figure 4.18 Character states mapping onto the MP tree of genus 60 Karlodinium ilh 15 character states and 9 taxa including 3 outgroups.

Vj

• ------.1\. LI T OF TABLES

Page Table 3.1 Reaction parameters for L U region amplification. 1-\

Table 4.1 Dinoflagellates isolated and established into clonal cultures 16 with their strains. isolat d date and location,

Table 4.:! Species from the four gener used in study . The LSU rR.."'A 30 gene sequences were obtained from GenBank with their origin. Strain designation and respective accession numbers.

Table -\. 3 Morphological characters and character states coded 111 this 36 study for the genus Karlodinium,

Table 4.4 Morphological characters and character states coded IJ1 thi s 39 study for genus Karenia,

Table 4,5 Morphological characters and character states coded III the 42 study for genus Takayama,

Table 4.6 Morphological characters and character states coded IJ1 thi s 44 study for the genus Gyrodinium.

Table 4.7 Di stribution of the character states among Karlodinium spp, 48 for the IS characters used in the character state evolution analysis,

Table 4,8 Distribution of the character states among Karenia spp, for the 4R 17 characters used in tlle character state evolution analysis.

Table 4,9 Distribution of the character states among Takayama spp, for 49 the IS characters used in the character state evolution analy is,

Table 4, I 0 Distribution of the character states among Gyrodinium spp. for 49 the 15 characters lIsed in the haraet r stale ~\'olutio n analysis,

V II

- _ . ---­ - '''',n. EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES, KARLODINlUMI KARENW TAKAYAMA! GYRODINlUM COMPLEX (D INOPHYCEAE)

Chow LuaD Jill

AB TRACT

The genera of 1\ r/vdillnilll. Karel/ia, TnkayalllG. GyrodilTlim are naked (.thecated) dinotlagell ates that have the potenti al to cause harmful algal blooms through Ihe production oftoxins or b} their accumulated biomass, which can affect co-occuning organisms and alter food·\Ieb dynami". In uus 'tudy. we examu" the phylogenetic lineage of I\arlodiu";",, I\a"

Key words: Naked dino fl agellates-, Karlodiniuml Karenia/Takayama/Gymnodiniffm complex, ribosomal RN A genes (rONA)

ABSTRAK

Genera Kor/odillmm, Karellfa Takayama, Gyrodil1l1l1J adnlah dinojlagt'/al yang mempwt)'oi kClIplI)'aOJl Un/Ilk nh!l~l'ebab~1J Icdakcl/1 olga dengan mel1ghasilkan 'oksm afau pengllmpu/all bioj/sf", yang boleh mempengarllhi nrgani.flllQ dan dinamik rankaifln makanal1 Do/am kajhUl ini, satu kajian fllogenclik Karlodinium, Karenia, Tak ayama dan Gyrodinium lelah dijo/anken dengan menjana pokok ji/ogene!ik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut. Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik do/am jilogeni Karlodinium dan Takayama, manakala genus Karenia mengungkapknn /iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOlloji/el,ic. Pemetaan ciri-ciri mv,.!ologi ke alas ji/ogen; memuJ} ukkan bahawa genus yang htlrbeza memiliki c;,.i-ciri marf%gi yang berbe::;a sebago; clrl-cin utama Unfllk pencctapan 5pesies, l\epllfUJen1 ini menunjuklwn bahawa Jwn;va b~be rapcJ ciri-d ri klasik (panj ang dan bel1{uk alur apikal, perpindahan sing/Jum. strllktllr sulkrl!l , kewujudan liang ventral) bermakna da/am konteks ji/ogenetik, Cirr-ciri lain .~epeni bemuk tpilwn da ll hipokon menunjllkkan varia ~' i yang tinggi dan tidak mempunyai nita; taksum:mlL Clri-cir; tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak.

Key wards: Naked dinojlagelal, KariodiniumiKareniaiTakayama/Gymnodll1 i,wl kompleks, gen rib(JSQmai RNA (rDNA)

V I) I

- . -----­ ~- 1.0 INTRODUCTION

The 'unanlloured' or 'naked' dinoflagellates lack cellulosic ampbiesmal plales. pecies of

unarmoured dinoflagellates are di tinguished by morphological characters such as size,

shape. po ilion and morphology of the cingulum (displacement andior overhang) and

sulcus. presence/absence of chloroplnsts and pyrenoids, shape and posi ti on of the nucleus,

shape of the apical furrow wben present. and possible surfaee structures. Observation of

other features, such as eyespots, species appendages is obviousl. also important.

Red tides or water di scoloration phenomena caused by an outbreak of a

heterotrophic dinoflagellate, Noctiluca scinlillans Kofoid and Swezy (1921 ) have been

fTequently observed in coastal areas, especially in eutrophic and enclosed bayments, for

more than several decades (Montani et al. 1998). Recurrent flsh kills have been detected

and were attributed to unamlO red, ichthyotoxic dinoflagellate. The e species make their

presence known in many ways because of the harm caused by their highly potent toxins.

The impacts of these phenomena include mass mortalities of wild and fanned fi sh and

sh Iltish, human intoxications or eVlln death from contaminated shellfish or fish,

alterations of marine trophic structure through adverse effects on larvae and other life

history stages of conunercial fisheri es species, and death of marine mammals. scabirds,

and other animals (Anderson 1995).

Correct and rapid detection of these hannful dinoflagellates speCles is important in

monitoring their dispersion throughout the world and minimizing fisb ery damage. The

main identification is based on microscopic examination, which requires considerable

taxonomic experience (K i 2005). Light microscope (LM) and Scanning El ectron

Microscope (SEM) arc commonly used to obseC\'e the morphological 'haracteristic of the

dinoflagellates_ How'ever, species delineation by traditional morphology-based

often presents challenges and provokes debate in dinoflagelJate systematic _ In order to

- . ------­ - ~- overcome the limitations of USIng morphological criteria alone to delineate pecles

boundaries, more comprehensive integrated approaches were incorporated, such as

molecular and physiological criteria tMuller et a!. 2007).

In this srudy. sampling was conducted in Kuehing vaters and naked dinoflagellates

were iso lated and 'stablish~d into clonal cultures. Species identification was performed by

using light microscopy ILM) and scanning lectton miCfCl copy (SEM). Clonal culrure

establi hed were used for genelIc characterization. Genomic DNA wa extracted and the

LSU rONA was amplified and sequenced. LSU TO A phylogenetic inference was

reconstructed by multiple sequence aligrunent and phylogenetic analyses. Analysis of

character state evolution .was performed by constructing the morphological characters

matrix. Morphological characters of Karlodinium, Karenia, Takayama and Gyrodinium

from literature description for each taxon was scored and mapped Ol1to the phylogeny. The

characters that generally used in taxonomic classification were chosen. The character

evolution of these species was then be elucidated.

The main objective of thi . study is to I!xamine the phylogendic lineage of nal...ed

dinoflagellates in relation to other phytoplankton species. The specifi c objectives are as

below:

I . To establish clonal cultures of naked dinoflagellates from Kuching w ater~;

2. To examine the morphology of naked dinoflagellates by using LM and SEM;

3. To infer the phylogenetic relationship of naked dinofla gellates especially

the Karlodiunim, Karenia, Takayama, Gyrodinium complex;

4. To determine the morphological characters those are of taxonomic value.

2

- • . ------­ ~~l . 2.0 LITERA TURE REVTEW

2.1 Naked dinoflageUates

Dinotlagellates lDillophyceae) are a large taxon onsi ling of numerous species which

inhabit different marine, brackish, and freshwater habitats. Dinoflagellates occur bOlh in

the water column. as a component of the plankton, and at t.he bouom of watcr bod ies. where they belung to the benthos. The taxon reveals an unmatched diversity of trophic types, from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites, each of

the categories being represented by numerous species (Bralewska & Witek 1995 ). The

variability within these classes is vast in many respects, but genetic, physiologic, and

morphological features are common to all of the species of the respective classes. All are

microscopic, the largest appearing to the naked eye as a minute globule. the smallest only

being apparent with the high power of a microscope. In size th ey range from 7 )J.m to 2 mm,

which is the size only occasionall y reached by Noctiliuca. the largest known dinoflagell ate.

A remarkable feature of dinoflagellates is their unique genome structure and gene

regulation. The nuclear genomt!s of these algae are of enomlOus size, lack Ilucleosomes,

and have permanently co ndensed chromosomes (Hackett 2004). Naked dinofl agellates has

been paid more attention as this taxonomy is artificial and misleading and many of the species cause extensive plankton blooms, fi sh kills and other hannful e,ents especiall y

fish ·killing species that are genera of Karlodinium J. Larsen. Karenill G. Hansen and

Moestrup, Takayama De Salas, Bolch, Botes and Hallegraeff and Gymnodinium Stein

(Daugbjerg 2000).

In this study. the genus Karlodin ium contains small. unarmoured, photosynthetic dinoflagellates that have chlocoplaSls with internal I~nticular pyrelloids. The cell covering is either with or without intracell ul ar plugs. The ventral epitbeca has a straight apical groove and a ventral pore. The main characters for distinguishing species between

3 Karlodinium were more likely the same with Karenia include position of nucleus, shape of

apical groove, sulcus structure and cingulum displacement ( teidinger et al. 2008). The

species in the Karlodinium complex such as Karl. an/arc /iellm de Salas (2008), Karl.

armiger Bergholtz, Daugbjerg et Moestrup (2006), Karl. au.flrale de Salas, Bo lch et

HaJlegraeff ~~005), Karl bal/al1lil1l1m de Salas (2008). Karl. coniClilll de Salas C:~008 ),

Karl. cormgallllll de Salas C~008 ) , Karl. decipiens de , alas et Laza-Martfnez (2008), Karl

miCl'lIm Larsen (2000) and Karl .. encflclim Larsen (2000).

The genus Karenia contains unarmoured, photosynthetic dinoflagellates, small to

medium size. with a straight apical groove on the ventral surface. Cell s are typically

vesiculate. The nucleus is round to oblong and without nuclear envelope chambers and a

nucleur capsule (S teidinger et a!. 2008). The main characters fo r distinguishing species

between Karenia incl ude the nuclear position, apical and sulcal groove details and relative

excavation of the hypotheca (Haywood et a!. 2004). The species in the Karenia complex

are K. asterichroma de Salas, Bolch et Hallegraeff (2004), K. bicuneijormis Botes, Sym et

Pitcher (2003). K. bidigitata Haywood et Steidi nger (2004), K. breris Hansen el Moestrup

(2000), K. brevisulca/a Hansen el Moestrup (2000), K. concordia Chang et Ryan (2 04), K.

crislala Botes, Sym et Pitcber (2003), K. digilala Yang, Takayama, Matsuoka et Hodgkiss

(20 01). K. IO llgicanalis Yang. Hodgkiss et Hansen (200 f), K. mikimoloi Daugbj~r g.

Hansen, Larsen, el Moestrup (2000), K. papilionacea I laywood et Sleidinger (2004), K

se/lijormis Haywood, Steidinger et MacKenzie (2004).

The genus Takayama has close affinities to the genera Karen io and Karlodinium. The

genus Takayama contains unarmoured, photosynthetic dinoflagellates with a sigmoid or "S,·

shaped apical groove. In certain species. the apical groove enciIcks the apex. Species are

either with sulcal intrusion or without sulcal intrusion into the epilheca and the cingulum is

descending (Steidinger et al. 2008). The main characters for distinguishing species 4

-.•~, PuJlll ~ MakllIIMt ,U de DII IDIlv.IITJ WALAYSIA SAItA'tIA&

between Takayama include position of nucleus, cell outline, p),Tenoid, and sulcus extension.

The species in the Takayama complex are T. acrotroclra Larsen, Flolch et HallegraefT

(2003), T. cladochroma Larsen, Bo lch e[ Hallegraeff (2003), T. helix de alas. Bolch.

Botes et Hallegraeff (2003), T. pulchella Larsen (1994), T. lasmanica de alas, Bolch [

HallegraefT(2003) and T. II/berell/ala de alas (::!008)

Tho;: species III GymJlodinium 'omplcx are G. bre\'e Davis (1948). G. carenatllm

Graham (1 943). G /uscum Ehrenberg F. lein (1878), G. micrum Braarud Taylor (199::!),

G mikimotoi Miyake et Kominami ex Oda (1935), G pulche/lum Larsen (1994), G.

sanguineum Hirasaka (1922), G. veneficum Ballantine (1956) and others.

5

.'-~-- 11 Toxoplasma gondll fa lctpa rum ' 00 IhermopniJa 100 elrahymena PYriformis p rotocenrrum mlcans P ·orocentrum meXJC.iinum Prnroccn ffum mInImum 5' PendJmelia catenBta 66 Hel@mcapsa sp Heterocaosa U", QueHa TOO Heu::mcapsa rotundala 86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium t'tollen 100 GYfTI.,od "tUm carenaru." GymnodInium fuscum 100 Gymnodinium palUSl re 55 Gymnodll'llum ct placldum 56 btl Gymnodin:um aureolum (USA) Gymnod niu m aureol um (Denmark) Gymnodinium chk>rophofum Gymnodinium ;mpudicu m Ka ren la breviS Kar enla ml kl mOtol (Oeomafk) 71 93 1'2 97 KaT enla IT1l k,moto. IJapan) Kanodl nJum ""arum 100 Akash wo sangulnea (USA) G5 100 Aka sOlwo sa ng Uinea (Canada) OJ .. Pondinium cin crum 100 Pe·lcflnlum pseu dolaeve Woloszynski a pseudop a l l J ~lns :00 Penc1'rllum Wilio. 83 100 Pendlnlum blpes 66 Alexandflum catenel (Austrohol

100 Alexand'lT1.Jm tamarense 95 Alexandnum catenella (USA) 91; ragilidlum 5ubgfobos um 52 P rO!OC 9 raHUi.'l reticula tum 97 Gonyaul"" s p l n~ e ra CerOllum tripos 88 Ceta !ium IInealum Ce:ra uum hJSUS 100 Amphldln lum carterae 100 Amphioln ium opc rculalum D lnop hy~i'S ac.umlnata

Fi gure 2.1: Phylogeny of major genera of dinoflagellates. tricl consensus of the J0 equally parsimonious trees obtained with the heuri stic search option in PAllP (source: Oaugbjerg 2000).

6

--=------.1l . 2.2 Harmful algal blooms

Toxic dinoflagellates, Karlodilll7im, Karenia, Takayama complex are arguabl y the

importWlt harmful algal bloom (HAS) species. based on the nwnber of species involved in

tOllic algal blooms and their exten ive geographical distri bution. In additiol) , these species

are responsible for paralytic shellfish poisoniog throughout the world. These organisms

pose an important problem in popUlation biology and taxonomy as well as a serious

e\:onomic and public health concern (Ki e l al. 2005),

Study of eutrophication conducted in Tolo Harbor showed that sun li ght was a

limiting factor to algal blooms (Lee & Arega 1999). However, many studies suggested that

nutrients in seawater could play more important roles in red tide occurrences (Hodgki 's &

Ho 1997), even though some other studies found that there were no strong correlations

between nutrient inputs and algal bloom intensity or/and frequ ency (Wear et aI . (984).

About 300 (7%) of the estimated 3,400-4, I 00 phytoplankton species have been

reported to produce "red tides" including diatoms, dinoflagellates, si licoflagellates,

prymnesiophytes, and raphidophytes ( ' ollmia (995), Most re el li ele species do not prodllce

harmful blooms. Only 60 -80 species (2%) ofthe 300 taxa are actually harmful or toxic as a

result of their biotoxins, physical damage, anoxia, irradiance reduction, nutritional

unsuitabili ty and others. Of these. flagellate species account for 90% and, among

flagellates, dinoflagellates stand out as a particularly noxious group. They account [o r 75%

(45 -60 taxa) of all hannful algal blooms (HABs) species. The exceptional importance of

dinoflagell ates is further evident from their pre-eminence among the species, perhaps 10­

12, primarily responsible for the current expansion and regional spreading of HAB

outbreaks in the sea (Anderson 1989),

7

~ . ------. ~, Many factors such as algal species presence or abundance, degree of flushing or

water exchange. weather conditions. and presence and abundance of grazers contribute to

the success of a given species at a gi ven point in time. Eutrophication is one of several

mechanisms by which harmful algae appear to be increasing in extent and duration in

many locations. Although important. it is not !he only explanation for blooms or toxic

utbreaks. Nutrient ertrichm~nt bas been strongly linked to stimulation of some harmful

species. but for others it has not been an apparent contributing facto r. The overall etTect of

nutrient over-enriclunent on harmful algal species i' clearly species speci fi c (Anderson et

a1. 2008).

During a HAB event, algal toxins can accumulate in predators and organIsms

higher up the food web. Toxins may also be present in ambient waters, where wave action

or human activities can create aerosols containing toxins and cell ular debris. Animals,

including humans, can thus be exposed to HAB-related toxins when they eat contaminated

seafood, have contact with contaminated water, or inhale contaminated aerosols. Given

that HAB events are becoming more frequent in the world's waters (Gli bert ct al. 2005). a

pressing need exists to understand, predict, and eventually mitigate the public-health

effects from these blooms (Lorraine 2006).

2.3 History of neurotoxic shellfish poisoning (NSP)

Neurotoxic shellfish pOlsomng (NSP) has been reported along the Gulf Coast in the

southeastern United States and eastern Mexico since the 1890s (Steidinger 1993) and NS P­

like s)mptoms have been reported by people eating shellfish from New Zealand (Ishida ct

al. 1996). Outbreaks of NSP have involved toxic oysters, clams. and other suspension

feeders that accumulate toxins during red tide HAB events. The toxins associated with

8

------", NSP are polyether compounds called brevetoxins (Baden 1989) produced by the

dinoflagellate Gymnodinium breve (formerly Plychodiscus brel'is and recentl y renamed

Karellia brevis [Daugbjerg et a!. 2000]) (Figure 2.2).

The acute symptoms ofN P are similar to those reported with ciguatera fish poisoning.

and include abdominal pain. oallSea, diarrhea, burning pain in the rectum. headache.

bradycardia, and dilated pupils. NSP victims have also reported temperature sensation

reversals, myalgia, vertigo. and ataxia (McFarren et a!. 1965). In addition to NSP,

brevetoxins can cause respiratory distress and eye irritation when individuals inhale sea

spray contaminated with these toxins (Music et al. 1973 ).

A variety of gastrointestinal tract and neurological symptoms were reported (Morris

1991). In addition, people with asthma show not only acute respiratory symptoms, but also

small changes in lung function immedi atel y after they spend even short periods of time on

the beach during Florida red tides when onshore winds cause aerosol exposures. Although

the underlying ecologic dynamics of these blooms and the ultimate source of nutrients

needed to produce such biomass are still under debate (Walsh & Steidinger 200 I), their

visibility from space has led to satellite-based method;; lor monitoring and prediction

(Stumpf et a1. 2003).

Satell ite imagery used to identifY areas that have undergone rapid changes in

chlorophyll concentrations, usually due to high growth, aggregation, or resuspension.

Because such temporal changes can al so be caused by bl ooms of non-toxic phytoplankton

species, suspected areas of K brevis red tide must be confinned with il7 situ measurements.

Following this confirmation, short-term predictions of bloom transport and landfall can be

computed using meteorologic forecasts to compute estimates of v.ind driven surface

currents to help managers decide where to obtain their nco t samples and how to prepar~ for

these blooms (Lorraine 2006).

9

------~. Figure 2.2: The dinoflagellate Karenia brevis, the causative organism of red tides on the West Florida shelf (David Patterson, Marine Biological Laboratory cited in Lorraine 2006).

2.4 Ribosom al RNA genes (rDNA) region

Many molecular techniques, such as alternative methods, have been developed to

discriminate between the morphological resemblances within the harmnll naked

dinoflagellates. These methods include isozyme patterns (CerobeUa et a1. 1988),

inunun ological propert ies (. ako et al. 1990), toxin prof(le (CembelJa et a\. 987) and more

recentl y used genetic ma 'eup (Destorobe et a1. 1992). Furthermore, with recent advances

in DNA sequencing technology, many DNA sequences are cunently re ealed and easily

available in the public database. With this knowledge, D equence-based genotyping is

a promising tool for the identification of harmful naked dinoflagellates (Ki et a\. 2005).

10

~ - ---- 11. 3.0 MATERlALS AND METHODS

3.1 ample eoUectiuD and clonal culture cstablishment

Ph}10plank"ton samplings were carried OUI stnned from August 2010 ill Semariang and

Sanlubong esruaries (Figure 3 J I b) using 20Jlll1 plankton nel. Liw samples \\'er~ brought

back to the laboratory for cell isolation. Cell isolation was arried OUI by using

micropipene t~chnique to establish clonal culture. Seawater (30 psu alinity) was use as [be

medium ase. Clonal cultures were established in SW II mediwn liwasaki 1961) and

maintained at 2S±0.SoC w1der a 12 hours of light and 12 hours of dark photocycJe. --­

••• Kuching

Figure 3.1: Map showing anrubong and 'emariang sampling site.

I I

- . ------1\ . 3.2 Species ide ntification

For LM. natural and cultured cells were fixed in 4% glutaraldehyde and examined wiLh an

Olympus IX5 1 microscope (Olympus. Melvi lle. NY, USA). Digital images were captured

using an attached cooled CCD camera (Soft Imaging System GmBH. Hamburg. Germ any).

Cell dimensions were determmed by measuring the dorsoventral diameter and

uan diameter of fixed cell , uslllg an eyepiece micrometer at mlignification '<-lOa.

Morphological characteristics. such as cell length (eL). cell widLh (CW) and cingulum

thickness were measured (Clui stine et a1. 2008). EM of the cell s were also been captured.

For SEM, the samples were came across six steps such as cell fixation, dehydration,

intermedium substitution, critical point dried, mounting and coating with gold. The cells

were fixed with 4% glutaraldehyde for I h and the fixative was di scarded. The cell

suspension was rinsed with 0.1 M cacodylate buff r for three ti mes. One percent OsO.

solution was added to cover the cell pellet and incubated. Cells were transferred into

polycarbonate (PC) membrane by mild filtration using vacuum manifold. Cells were rinsed

three times with dl·Hl to remove salt and fixatives, dehydrated in a graded series of ethyl

(30-100%) and filter-mounted to a stub. TIlen, the specimens were treated wiLh

intermedium sub stitution by using graded baths EtOH : Amyl acetale of (75:25, 50:50,

25: 75) perceillage and finally transferred into 100 percent amyl ac tale fo ll o\-\ ed be cri tical

point drying process (CPD). The samples were stored in a va uum desiccator. Specimen

were coated with gold using a JFC - 1600 (.TEaL, Japan) before observed under a scanning

electron microscope (.lEaL, JSM·65 10, Japan). Average celJ dimensions were calculated

from measurements made on 20 cells.

12

------~. 3.3 Genomic DNA extraction

Genomic extraction was carried out according to Leaw et al. (2009). Approximately 125 to

150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5

minutes) for genomic extraction. The cell pell et was lyses by adding of:! x CTA.B (Cetyl­

trimetyl ammonium bromide) buffer consists 0.02 M EDTA. 0.06 M cr AB, 0.1 M Tris­

Base. 1.4 M NaCI and I ml of 2 - ~ - 01e rcaptoetbano l l.\li th addition of 5 fll Proteinase K

(20mg/01I; QIAGE. ). The mixture was incubated at 65°C for 10 minute before extracted

on e with chloroform-isoamyl alcohol (24: 1). The samples were subsequently extracted

using equal volume standard phenol-chloroform procedures. DNA was extracted by

centrifugation at 10000 rpm fo r 10 min. Repeated the steps by using phenol chloroform

isoamyl followed by chloroform isoamyl again. The DNA was precipitated by adding

equal volume of absolute ethanol and 25 fll of3 M sodi um acetate (N aOAc, pH 5.0). The

samples were then stored in _20DC for 3 hours. Mixture was then centrifuged at 13000 rpm

for 10 min and the D A pellet was rinsed with 70% ethanol followed by centrifugation

again. DNA pell et was allowed In ir dry at room temperatllre. The DNA pellet was then

dissolved in 50 ).11 0 IT buffer (10 01.\1 Tris-HCl. p I-l 7 .~; I mM EDTA. pII 8.0).

3.4 Am plification and sequencing of rDNA

Amplification of the large subunit (LSU) ribosomal Rt"lA gene was carried out by using

primer pair, DIR. 5'- ACC CGC TOA ATT TAA OCA TA - 3' and D3Ca, 5' - ACO

AAC OAT TOC ACO TCA 0 - 3'(Scholin et al. 1994). The PCR master mix contained of

I x peR buffer tP romega. Madi son. WI. U:A), 2.5 Uh\1 MgCI". 0.4 mM of each dAT P,

dTIP, dCTP and dGT ]> (QIAGEN, OmbH, Hilden, Germany). 0.02 ~ I M of each primer,

13

-,------~ ~.