evolutionary lineage of naked harmful …
TRANSCRIPT
EVOLUTIONARY LINEAGE OF NAKED HARMFUL DINOFLAGELLATES KARLODINIUMIKARENIAITAKA YAMAI
GYRODINIUM COMPLEX (DINOPHYCEAE)
Chow Luan Jia
Bachelor of Science with Honours (Resource Biotechnology)
2011
~ sa u idlAUl1fllu lNWM1I MampLArM SAMWAt
EVOLUTIONARY LINEAGE OF NAKED HARMF L DU OFLAGELLATES KARLODINIUMI KARENAI TAKA yAftLV 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 Ttchnology lInicroIlY Malaysia Sanlak
2011
middott bullbull bull
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 qual ification to this or any other univ rsity
nr insti tution of higher Ieaming
(~~ CHOW LUA JIA
Resource Biotechnology Programme
Facu lty of Resou rce Science and Technology
Univers ity 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 thankgt to the Sarawak Fisheries Department for the
acccssibility to the sampling site
Great appreciation to the following individuals for the ir 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 office rs 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 foundation for this study
This project was supported by MOSTI eScience Fund to Dr Leaw
TABLE OF CONTENTS
Page
ACKNOWLEDGEME TS
TABLE OF COolTENT ii
LI T OF FIGURE
LIST OF TABLES Vll
LI T OF ABBREVIATIONS iv
ABSTRACT viii
ABSTRAK viii
10 LITROD CTION
20 LITERATURE REVIEW 3
21 Naked dinoflagellates 3
22 Harmful alga blooms 7
23 History of neurotoxic shellfi sh poisoning (NS) 8
24 Ribosomal RNA genes (rONA) region 10
30 MATERIA LS AND METHODS I I
31 Sample ollection and clonal cul rurc clablishment II
32 Species identificat ion 12
33 Genomic 0 A extraction J3
34 Amplification and sequencing of rONA 13
35 Phylogenetic analyses 14
35 1 Sequence analysis and taxon ampling 14
35 2 LSU phylogenetic analysis 15
353 Matrix construction for morphologica l middotmiddotlaraclers J5
11
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16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
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LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
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Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
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Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
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16
30
36
39
42
44
48
4R
49
49
V II
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EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
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10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
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overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
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20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
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22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
12 primarily responsible for the current expansion and regional spreading of HAB
outbreaks in the sea (Anderson 1989)
7
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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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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
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NSP are polyether compounds called brevetoxins (Baden 1989) produced by the
dinoflagellate Gymnodinium breve (formerly Plychodiscus brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
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30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
~ sa u idlAUl1fllu lNWM1I MampLArM SAMWAt
EVOLUTIONARY LINEAGE OF NAKED HARMF L DU OFLAGELLATES KARLODINIUMI KARENAI TAKA yAftLV 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 Ttchnology lInicroIlY Malaysia Sanlak
2011
middott bullbull bull
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 qual ification to this or any other univ rsity
nr insti tution of higher Ieaming
(~~ CHOW LUA JIA
Resource Biotechnology Programme
Facu lty of Resou rce Science and Technology
Univers ity 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 thankgt to the Sarawak Fisheries Department for the
acccssibility to the sampling site
Great appreciation to the following individuals for the ir 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 office rs 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 foundation for this study
This project was supported by MOSTI eScience Fund to Dr Leaw
TABLE OF CONTENTS
Page
ACKNOWLEDGEME TS
TABLE OF COolTENT ii
LI T OF FIGURE
LIST OF TABLES Vll
LI T OF ABBREVIATIONS iv
ABSTRACT viii
ABSTRAK viii
10 LITROD CTION
20 LITERATURE REVIEW 3
21 Naked dinoflagellates 3
22 Harmful alga blooms 7
23 History of neurotoxic shellfi sh poisoning (NS) 8
24 Ribosomal RNA genes (rONA) region 10
30 MATERIA LS AND METHODS I I
31 Sample ollection and clonal cul rurc clablishment II
32 Species identificat ion 12
33 Genomic 0 A extraction J3
34 Amplification and sequencing of rONA 13
35 Phylogenetic analyses 14
35 1 Sequence analysis and taxon ampling 14
35 2 LSU phylogenetic analysis 15
353 Matrix construction for morphologica l middotmiddotlaraclers J5
11
--------- shy ~
-------
16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
-- ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
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 qual ification to this or any other univ rsity
nr insti tution of higher Ieaming
(~~ CHOW LUA JIA
Resource Biotechnology Programme
Facu lty of Resou rce Science and Technology
Univers ity 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 thankgt to the Sarawak Fisheries Department for the
acccssibility to the sampling site
Great appreciation to the following individuals for the ir 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 office rs 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 foundation for this study
This project was supported by MOSTI eScience Fund to Dr Leaw
TABLE OF CONTENTS
Page
ACKNOWLEDGEME TS
TABLE OF COolTENT ii
LI T OF FIGURE
LIST OF TABLES Vll
LI T OF ABBREVIATIONS iv
ABSTRACT viii
ABSTRAK viii
10 LITROD CTION
20 LITERATURE REVIEW 3
21 Naked dinoflagellates 3
22 Harmful alga blooms 7
23 History of neurotoxic shellfi sh poisoning (NS) 8
24 Ribosomal RNA genes (rONA) region 10
30 MATERIA LS AND METHODS I I
31 Sample ollection and clonal cul rurc clablishment II
32 Species identificat ion 12
33 Genomic 0 A extraction J3
34 Amplification and sequencing of rONA 13
35 Phylogenetic analyses 14
35 1 Sequence analysis and taxon ampling 14
35 2 LSU phylogenetic analysis 15
353 Matrix construction for morphologica l middotmiddotlaraclers J5
11
--------- shy ~
-------
16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
-- ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
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 thankgt to the Sarawak Fisheries Department for the
acccssibility to the sampling site
Great appreciation to the following individuals for the ir 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 office rs 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 foundation for this study
This project was supported by MOSTI eScience Fund to Dr Leaw
TABLE OF CONTENTS
Page
ACKNOWLEDGEME TS
TABLE OF COolTENT ii
LI T OF FIGURE
LIST OF TABLES Vll
LI T OF ABBREVIATIONS iv
ABSTRACT viii
ABSTRAK viii
10 LITROD CTION
20 LITERATURE REVIEW 3
21 Naked dinoflagellates 3
22 Harmful alga blooms 7
23 History of neurotoxic shellfi sh poisoning (NS) 8
24 Ribosomal RNA genes (rONA) region 10
30 MATERIA LS AND METHODS I I
31 Sample ollection and clonal cul rurc clablishment II
32 Species identificat ion 12
33 Genomic 0 A extraction J3
34 Amplification and sequencing of rONA 13
35 Phylogenetic analyses 14
35 1 Sequence analysis and taxon ampling 14
35 2 LSU phylogenetic analysis 15
353 Matrix construction for morphologica l middotmiddotlaraclers J5
11
--------- shy ~
-------
16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
-- ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
TABLE OF CONTENTS
Page
ACKNOWLEDGEME TS
TABLE OF COolTENT ii
LI T OF FIGURE
LIST OF TABLES Vll
LI T OF ABBREVIATIONS iv
ABSTRACT viii
ABSTRAK viii
10 LITROD CTION
20 LITERATURE REVIEW 3
21 Naked dinoflagellates 3
22 Harmful alga blooms 7
23 History of neurotoxic shellfi sh poisoning (NS) 8
24 Ribosomal RNA genes (rONA) region 10
30 MATERIA LS AND METHODS I I
31 Sample ollection and clonal cul rurc clablishment II
32 Species identificat ion 12
33 Genomic 0 A extraction J3
34 Amplification and sequencing of rONA 13
35 Phylogenetic analyses 14
35 1 Sequence analysis and taxon ampling 14
35 2 LSU phylogenetic analysis 15
353 Matrix construction for morphologica l middotmiddotlaraclers J5
11
--------- shy ~
-------
16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
-- ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
-------
16 40 RESULTS
41 Algal cul tures establ i hed 16
42 Species identification 16
411 Protocerarium rericuiatum 17
42 2 Prorocentrum rhathymum 19
423 GyrodmiulII illslriarum 21
)424 Alexandrilllll sp - ~
425 Akashill 0 sal1guinea 24
426 ochlodinium cf p ofykrikoides 25
42 7 Prorocentrum sigmoides 26
428 Karlodinium veneficwn 27
43 Genomic DNA extracti on amplification and purification 29
44 Taxon sampling 30
45 Phylogenetic inferences of naked dinoflagellates 31
451 Karlodinium phylogeny 31
452 Karenia phylogeny 32
45 3 Takayama phylogeny 33
454 Gyrodinium phylogeny 34
46 Morphological traits 35
47 Matrices constructed for character state evolution 47
48 Character state evolution 50
481 Character state evolution of Karlodiniwn 50
482 Character state evolution of Karenia 53
483 Character state evolution of Takavama 56
484 Character state evolution of Gyrodinillm 58
50 DISCUSSION 60
60 CO CLU ION 69
70 REFERENCES 70
III
-- ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
----
LIST OF ABBREVI TJONS
N P
BLAST
EM
LM
HAB
rRNA
CPO
Neurotoxic Shellfi sh Poisoning
Basic local aJigIunent search tool
Scanning Electron Microscope
Light Microscope
Harmful algal bloom
Ribosomal genes
Cri tical Point Dried
IV
~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
------
Figure 21
middot )Igur~ _ _F
Figure 31
F igure -t 1
Figure 42
Figure 43
Figure 44
Figure 45
Figure 46
Figure 47
Figure 48
Figure 49
Figure 410
Figure 4 12
Ll T OF FIGURE
Phylogeny of major g nera of dinoflag Hates trict consensus of the 10 equally parsimonious trees obtained with the heuristic eareh option in P UT (Oaug~i erg ~OOO)
P ge
6
The dinoflagellate Aarenia brevis the causative organism of red tides on tb West Florida sbelf (Daid Partersoo 1arille Biological Laboratory cited in Lorraine 2006)
10
Map hoing antubong and Semariang sampling site 13
Light and scanning electron micrographs reticularum fro m Semariang Sarawak
of Preemi lln 18
Light and scaMing electron micrographs rhahymum from Semariang Sarawak
of Prorocenrrum 20
Light micrographs of Gymnodinium inslriarum from Santubong Sarawak
21
ScaMing electron micrographs of Gymnodinium insriaurn from Saotubong Sarawak
22
Light and scanning electron micrographs from Semariaog Sarawak
of Alexandrium sp 23
Light and autofluorescence micrographs of Akashiwo sangllinea from Semariang Samwak
24
Light aod autofluorescence micrographs of Cochlodiniwn polykrikoides from Semariang Sarawak
cf 25
Light and autofluorescence micrographs sigmoides fro m Selllariang Sarawak
of Prorocentrum 26
Scanning electron micrographs of Karlodinium Johore
venejiculIl from 28
Negative image of gel for purified PCR products of LS rONA 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
29
MP tree of Kariodin ill lll with tree length 01 61 2 evolutionary steps and bootstrap alue of 1000 The consistenc) index (el) was 08284 and retention index IRJ ) - 06602
31
v
~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
Figure 412
Figure 413
Figure 414
Figure middotU 5
Figure 416
Figure 417
Figure 418
MP tree of Karenia with tree length of 480 evolutionary steps and bootstrap value of 1000 The consistency index (CI) was 08583 and retention index (Rl) =05613
32
MP tree of Takayama with tree length of 392 e olutionary steps and bootstrap yulue of 000 The consistency index (CI) was 09337 and retention index (RI) =07204
33
vIP tree of Gyrodinillm with tree length of 745 evolutionary steps and bootstrap value of 1000 n1e consistency index (Cl ) was 08389 and retention index tID) =053-19
34
Character states Karlodinillln with outgroups
mapping onto the MP 15 character states and I I
tree of genus taxa including 3
52
Character states mapping onto the MP tree of genus Karenia with 17 character states and 9 taxa including 3 outgroups
55
Character states mapping onto the MP tree of genus Takayama with 15 character states and 8 taxa including 3 outgroups
57
Character states Karlodinium ilh outgroups
mapping onto the MP tree of genus 15 character states and 9 taxa including 3
60
Vj
bull --------- 1
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
Table 31
Table 41
Table 4
Table -3
Table 44
Table 45
Table 46
Table 47
Table 48
Table 49
Table 4 I 0
LI T OF TABLES
Reaction parameters for L U region amplification
Dinoflagellates isolated and established into clonal cultures with their strains isolat d date and location
Species from the four gener used in study The LSU rRA gene sequences were obtained from GenBank with their origin Strain designation and respective accession numbers
Morphological characters and character states coded 111 this study for the genus Karlodinium
Morphological characters and character states coded IJ1 thi s study for genus Karenia
Morphological characters and character states coded III the study for genus Takayama
Morphological characters and character states coded IJ1 thi s study for the genus Gyrodinium
Distribution of the character states among Karlodinium spp for the IS characters used in the character state evolution analysis
Distribution of the character states among Karenia spp for the 17 characters used in tlle character state evolution analysis
Distribution of the character states among Takayama spp for the IS characters used in the character state evolution analy is
Distribution of the character states among Gyrodinium spp for the 15 characters lIsed in the haraet r stale ~olution analysis
Page
1-
16
30
36
39
42
44
48
4R
49
49
V II
_ - --- shy - n
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
EVOLUTIONARY LINEAGE OF NAKED H RMFUL DINOFLAGELLATES KARLODINlUMI KARENW TAKAYAMA GYRODINlUM COMPLEX
(D INOPHYCEAE)
Chow LuaD Jill
AB TRACT
The genera of 1 rvdillnilll Karelia TnkayalllG GyrodilTlim are naked (thecated) dinotlagellates that have the potential to cause harmful algal blooms through Ihe production of toxins or b thei r accumulated biomass which can affect co-occuning organisms and alter foodmiddotIeb dynami In uus tudy we examu the phylogenetic lineage of Iarlodiu Ialtma Takayama GJr()dinllllll complex by mapping the morphological characters ontO Ihe pbylogenetic (fee Using LSU rRNA gene sequences inferences AUTlodinium and Takayama phylogenies each revealed two monophyletic groups respective ly whereas Kfenia revealed wec monophyletic groups and GyrodiniulI revealed only one monophyletic group Character mapping onto the LSU phyJogeny reveaJed that d ifferent genera possess d ifferen t morpho logical cbaracters as the major morphological traits fo r species delineation It appears that only certain classic morphological features (length and sbaped of apical groove cingUlum displacement sulcus structure present of ventraJ pore) are of phylogenetic signiicance Tbe other cbaracters sucb as the shape of epicone and hypocone somehow show high levels of variety and possess no taxonomic diagnost ic value in the genera These characters did not strongly support any grouping of taxa and appeared to bave evolved in a random fashi on
Key words Naked dino fl agellates- Karlodiniuml KareniaTakayamaGymnodini ffm complex ribosomal RN A genes (rONA)
ABSTRAK
Genera Korodillmm Karellfa Takayama Gyrodil1l1l1J adnlah dinojlagtal yang mempwt)oi kClIplI)aOJl UnIlk nhl~lebab~1J Icdakcl1 olga dengan mel1ghasilkan oksm afau pengllmpuall biojsf yang boleh mempengarllhi nrganiflllQ dan dinamik rankaifln makanal1 Doam kajhUl ini satu kajian fllogenclik Karlodinium Karenia Tak ayama dan Gyrodinium lelah dijoanken dengan menjana pokok jiogeneik dan seterusnya memetakan ciri-ciri m01fologi ke alas pokok jilogenetik fersebut Analisis filogeni berasakon gen LSU rRNA mengungkapkan duo kumpulan monofiletik doam jilogeni Karlodinium dan Takayama manakala genus Karenia mengungkapknn iga kum ulan monofiletik dan Gyrodinium satu kwnpulan mOllojielic Pemetaan ciri-ciri mvologi ke alas jiogen memuJ ukkan bahawa genus yang htlrbeza memiliki ci-ciri marfgi yang berbea sebago clrl-cin utama Unfllk pencctapan 5pesies lepllfUJen1 ini menunjuklwn bahawa Jwnva b~berapcJ ciri-d r i klasik (panj ang dan bel1uk alur apikal perpindahan singJum strllktllr sulkrll kewujudan liang ventral) bermakna daam konteks jiogenetik Cirr-ciri lain ~epeni bemuk tpilwn dall hipokon menunjllkkan varia~ i yang tinggi dan tidak mempunyai nita taksummlL Clri-cir tersebullidak menyokong sebarang pengelompokan (axa dan berubah secara rawak
Key wards Naked dinojlagelal KariodiniumiKareniaiTakayamaGymnodll1 iwl kompleks gen rib(JSQmai RNA (rDNA)
V I) I
- ----- shy ~-
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
10 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 presenceabsence of chloroplnsts and pyrenoids shape and posi tion 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 kill s have been detected
and were attributed to unamlOred 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 shell fish 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 fisbery damage The
main identification is based on microscopic examination which requires considerable
taxonomic experience (K i 2005) Light microscope (LM) and Scanning Electron
Microscope (SEM) arc commonly used to obseCe the morphological haracteristic of the
dinoflagellates_ However species delineation by traditional morphology-based taxonomy
often presents challenges and provokes debate in dinoflagelJate systematic _ In order to
- --------shy - ~-
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
overcome the limitations of USIng morphological criteria alone to delineate pecles
boundaries more comprehensive integrated approaches were incorporated such as
molecular and physio logical 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 Ixamine the phylogendic lineage of naled
dinoflagellates in relation to other phytoplankton species The specific objectives are as
below
I To establish clonal cultures of naked dinoflagellates from Kuching water~
2 To examine the morphology of naked dinoflagellates by using LM and SEM
3 To infer the phylogenetic relationship of naked dinoflagellates especially
the Karlodiunim Karenia Takayama Gyrodinium complex
4 To determine the morphological characters those are of taxonomic value
2
bull ~l - ------ shy ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
20 LITERA TURE REVTEW
21 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 the bouom of watcr bod ies
where they belung to the benthos The taxon reveals an unmatched d iversity of trophic
types from pure autotrophs through mixotrophs and pure heterotrophs 10 parasites each of
the categories being represented by numerous species (Bralewska amp 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 they range from 7 )Jm 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 genomts of these algae are of enomlOus size lack Ilucleosomes
and have permanently condensed chromosomes (Hackett 2004) Naked dinoflagellates 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 eents especiall y
fish middotkilling species that are genera of Karlodin ium 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 intrace ll 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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
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 anarc iellm de Salas (2008) Karl
armiger Bergholtz Daugbjerg et Moestrup (2006) Karl auflrale de Salas Bolch et
HaJlegraeff ~~005) Karl balal1lil1l1m de Salas (2008) Karl coniClilll de Salas C~008 )
Karl cormgallllll de Salas C~008) Karl decipiens de alas et Laza-Martfnez (2008) Karl
miCllIm 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 include the nuclear posi tion 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 brevisulcaa 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
(2001) K IOllgicanal is Yang Hodgkiss et Hansen (200 f) K mikimoloi Daugbj~rg
Hansen Larsen el Moestrup (2000) K papilionacea I laywood et Sleidinger (2004) K
selijormis 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 Smiddot
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
-bull ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
PuJlll ~ MakllIIMt U de DII IDIlvIITJ WALAYSIA SAItAtIAamp
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 Bolch 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 IIberellala de alas (008)
Tho species III GymJlodinium omplcx are G bree 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 pulchelum Larsen (1994) G
sanguineum Hirasaka (1922) G veneficum Ballantine (1956) and others
5
11-~--
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
Toxoplasma gondll
PlasmodIum fa lctparum Tetrahymena IhermopniJa 00
100 elrahymena PYriformis p rotocenrrum mlcans P middotorocentrum meXJCiinum
Prnroccn ffum mInImum PendJmelia catenBta 5
66 Helmcapsa sp
Heterocaosa UQueHa TOO Heumcapsa rotundala
86 SenpPslella sp 100 ScnppStell8 trocholdea val aClculrfera 100 GymnodInium ttollen 100 GYfTIod tUm carenaru
GymnodInium fuscum100
Gymnodinium palUSlre 55 Gymnodllllum ct placldum 56 btl Gymnodinum aureolum (USA)
Gymnod nium aureol um (Denmark)
Gymnodinium chkgtrophofum Gymnodinium mpudicum Karenla breviS Karenla mlklmOtol (Oeomafk) 71 93
12 KaTenla IT1lkmoto IJapan) 97 KanodlnJum arum
100 Akash wo sangulnea (USA ) 100 Aka sOlwo sa ngUinea (Canada)
OJ G5
Pondinium cincrum 100 Pemiddotlcflnlum pseudolaeve
Woloszynskia pseudopa l lJ~lns
00 Penc1rl lum Wilio 83
100 Pendlnlum blpes66
Alexandflum catenel (Austrohol
AlexandlT1Jm tamarense100 Alexandnum catenella (USA) 95
91 ragilidlum 5ubgfobosum 52 P rOOC9 raHUil reticula tum
97 Gonyaul spln~era
CerOllum tripos88
Cetaium IInealum
Cerauum hJSUS
100 Amphldlnlum carterae 100 Amphiolnium opcrculalum
Dlnophy~iS acumlnata
Figure 21 Phylogeny of major genera of dinoflagellates tricl consensus of the J0 equally parsimonious trees obtained with the heuristic search option in PAllP (source Oaugbjerg 2000)
6
--=- -- -- - 1l
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
------
22 Harmful algal blooms
Toxic dinoflagellates Karlodilll7im Karenia Takayama complex are arguably 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
eonomic and public health concern (Ki e l al 2005)
Study of eutrophication conducted in Tolo Harbor showed that sun light was a
limiting factor to algal blooms (Lee amp Arega 1999) However many studies suggested that
nutrients in seawater could play more important roles in red tide occurrences (Hodgki s amp
Ho 1997) even though some other studies found that there were no strong corre lations
between nutrient inputs and algal bloom intensity orand frequency (Wear et aI (984)
About 300 (7) of the estimated 3400-4 I 00 phytoplankton species have been
reported to produce red tides including diatoms dinoflagellates si licoflagellates
prymnesiophytes and raphidophytes ( ollmia (995) Most reel liele species do not prodllce
harmful blooms Only 60-80 species (2) of the 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 10shy
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
- -----
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 on ly explanation for blooms or toxic
utbreaks Nutrient ertrichm~nt bas been strongly linked to stimu lation 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 worlds 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)
23 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 Pshy
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 brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
- -----
NSP are polyether compounds called brevetoxins (Baden 1989) produced by the
dinoflagellate Gymnodinium breve (formerly Plychodiscus brelis and recently renamed
Karellia brevis [Daugbjerg et a 2000]) (Figure 22)
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 immediately 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 amp Steidinger 200 I) their
visibi lity 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 also 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 vind 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 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
Figure 22 The dinoflagellate Karenia brevis the causative organism of red tides on the West Florida she lf (David Patterson Marine Biological Laboratory cited in Lorraine 2006)
24 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)
inununological propert ies ( ako et al 1990) toxin prof(le (CembelJa et a 987) and more
recently used genetic ma eup (Destorobe et a1 1992) Furthermore with recen t 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
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
-
30 MATERlALS AND METHODS
31 ample eoUectiuD and clonal culture cstablishment
Ph10plankton 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 cu lture Seawater (30 psu alinity) was use as [be
medium ase Clonal cultures were established in SW II mediwn liwasaki 1961) and
maintained at 2Splusmn0SoC w1der a 12 hours of light and 12 hours of dark photocycJe
--shy
bullbullbull
Kuching
Figure 31 Map showing anrubong and emariang sampling site
I I
----- - 1
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
- - ------
32 Species identification
For LM natural and cultured cells were fixed in 4 glutara ldehyde 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 Germany)
Cell dimensions were determmed by measuring the dorsoventral diameter and
uan diameter of fixed cell uslllg an eyepiece micrometer at mlignification lt-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 01 M cacodylate buff r for three times One percent OsO
solution was added to cover the cell pellet and incubated Cells were transferred into
polycarbonate (PC) membrane by mi ld filtration using vacuum manifold Cells were rinsed
three times with dlmiddotHl 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 substitution by using graded baths EtOH Amyl acetale of (75 25 5050
25 75) perceillage and finally transferred into 100 percent amyl ac tale fo llo- ed be cri tical
point drying process (CPD) The samples were sto red in a va uum desiccator Specimen
were coated with gold using a JFC - 1600 (TEaL Japan) before observed under a scanning
electron microscope (lEaL JSMmiddot65 10 Japan) Average celJ dimensions were calculated
from measurements made on 20 cells
12
~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~
33 Genomic DNA ex traction
Genomic extraction was carri ed out according to Leaw et al (2009) Approximate ly 125 to
150 ml of mid-exponential batch culture were harvested by centrifugation (3 000g for 5
minutes) for genomic extraction The cell pellet was lyses by adding of x CT AB (Cetylshy
trimetyl ammonium bromide) buffe r consists 002 M EDTA 006 M cr AB 01 M Trisshy
Base 14 M NaCI and I ml of 2 - ~ -01ercaptoetbanol lli th addition of 5 fll Proteinase K
(20mg01I QIAGE ) The mixture was incubated at 65degC 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 50) 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 pe ll et was allowed In ir dry at room temperatllre The DNA pellet was then
dissolved in 50 )11 0 IT buffer (10 011 Tris-HCl pI-l 7 ~ I mM EDTA pII 80)
34 Am plification and sequencing of rDNA
Amplification of the large subunit (LSU) ribosomal RtlA 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 Madison WI UA) 25 Uh1 MgCI 04 mM of each dAT P
dTIP dCTP and dGT ]gt (QIAGEN OmbH Hilden Germany) 002 ~IM of each primer
13
-- -----~ ~