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ECOLOGY AND RAPID DETECTION OF DIATOM Pseudo-nitzschia (BACILLARIOPHYCEAE) IN KUCHING ESTUARIES Wang Chuan Yu QK 569 DS4 W146 1111 Bachelor of Science with Honours (Aquatic Resource Science and Management) 2012

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Page 1: ECOLOGY AND RAPID DETECTION OF DIATOM Pseudo-nitzschia ... and Rapid... · Ecology and rapid detection of diatom Pseudo-nitzschia (Bacillariaphyceae) at Kuching estuaries . Wang Chuan

ECOLOGY AND RAPID DETECTION OF DIATOM Pseudo-nitzschia (BACILLARIOPHYCEAE)

IN KUCHING ESTUARIES

Wang Chuan Yu

QK 569 DS4 W146 1111

Bachelor of Science with Honours (Aquatic Resource Science and Management)

2012

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Pusat Khidmat Maklumat Akademik UNlVEltSm MALAYSIA SAltAWAK

Ecology and rapid detection of diatom Pseudo-nitzschia (Bacillariophyceae) in Kuching estuaries

P.KHIDMAT MAKLUMAT AKADI!MIK

111111111 r0I1II11 I11I11 1000235659

Wang Chuan Yu

A project report submitted in partial fulfillment of the Final Year Project (STF 3015)

Supervisor: Dr Lim Po Teen Co-supervisor: Dr Leaw Chui Pin

Aquatic Resource Science and Management Department of Aquatic Science

Faculty of Resource Science and Technology University Malaysia Sarawak

(2012)

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DECLARATION

I hereby declare that this thesis is based on my original work except for quotations and

citation, which have been duty acknowledged. I also declare that it has not been

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

institutions.

Wang Chuan Yu

Aquatic and Resource Science and Management Programme

Department of Aquatic Science

Faculty ofResource Science and Technology

Universiti Malaysia Sarawak

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ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr Lim Po Teen and my co-supervisor Dr Leaw Chui

Pin for their advices, support, assistance and encouragement during the course of this Final

Year Project and completion of thesis. In addition, I would like to thanks aU the lecturers

from Aquatic science Department for their guidance and advices .

Special thanks to the !BEC Molecular Laboratory seniors Teng Sing Tung, Hii

Kieng Soon, Tan Toh Hii and Lim Hong Chang for their assistances along the way and

guidance in handling the lab apparatus. Thanks to Mr. Zaidi, Mr. Azlan, and Mdm Ting for

apparatus preparation in field sampling and laboratory work.

Lastly, this thesis is dedicated to my parents for their lifetime support and

encouragements.

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Pusat Khidmat MakJumlt Akademik UNlVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF FIGURES

LIST OF TABLES ABSTRACT

ABSTRAK 1.0 INTRODUCTION

2.0 LITERA TURE REVIEWS 2.1 Harmful Algal Bloom

2.2 The genus Pseudo-nitzschia

2.3 Amnesic Shellfish Poisoning (ASP)

2.4 Factors influence the growth and distribution of Pseudo-nitzschia

2.4.1

2.4.2 2.4.3

2.4.4

Environmental factor

Temperature and irradiance Salinity

Nutrients

2.5 Whole- cell Fluorescence In-situ Hybridization (FISH)

3.0 MATERIALS AND METHODS

3.1

3.2

3.3

3.4 3.5

3.6

3.7 3.8

3.9

3.10

Sampling Site

In-situ Measurement

Cell Counting

Cell Isolation

Medium Preparation

Acid Wash of Pseudo-nitzchia Cultures Sample & Samples Observation Nutrients Analysis

Phylogenetic analyses In silico oligonucleotide probe design

FISH protocol

4.0 RESULTS AND DISCUSSION 4.1 Distribution of Pseudo-nitzschia in Kuching estuary

4.1.1

4.1.2 4.1.3 4.1.4 4.1 .5

Temporal distribution of Pseudo-nitzschia in Samariang & Santubong Physical parameters Macronutrients Average Rainfall two day before sampling Physical - chemical factors affecting distribution Pseudo-nitzschia

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4.2 Algal cultures 28 4.3 Morphological Observation of Pseudo-nitzschia cultures

4.3.1 Morphology of Pseudo-nitzschia brasiliana 29 4.3.2 Morphology of Pseudo-nitzschia pungens 29 4.3.3 Morphology of Pseudo-nitzschia caciantha 30 4.3.4 Morphology ofPseudo-nitzschia circumpora 30

4.4 Phylogenetic inferences 33 4.5 In-silico probe design 36

4.5.1 Pseudo-nitzschia circumpora species specific probe 38 design

4.5.2 Pseudo-nitzschia caciantha species specific probe 41 design

4.5.2 Pseudo-nitzschia pungens species specific probe 44 design

4.6 Optimization of Species-specific P. pungens probe 47

5.0 CONCLUSION 51

6.0 REFERNCES 52

Appendices A P. circumpora potential probes selection by ARB package 57

strains and GenBank accession numbers used in this study

parameters data obtained from Santubong and Samariang estuaries

B P. caciantha potential probe selection by ARB package 61 C Signature region predicted in P. caciantha 62 0 P. pungens potential probe selection by ARB package 63 E LSU RNA genes (DI-D3) sequences of of Pseudo-nitzschia 64

F Raw data of cell abundance and in situ physico-chemical 65

111 ....

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,..

LIST OF ABBREVIATIONS

ASP Amnesic Shellfish Poisoning

BI Bayesian inference

BLAST Basic Local Alignment Search Tool

CBC Compensatory base change

DA Domoic Acid

EDTA Ethylenediamine-Tetraacetic Acid

FISH Fluorescence In Situ Hybrdisation

ITS1 Internal transcribed spacer I

LSUrRNA Large Subunit Ribosomal Ribonucleic Acid

ML Maximum Likelihood

MP Maximum Parsimony

N02- Nitrite

N03-, Nitrate

NT Nucleotide

SWII Seawater II medium I

Si02 Silicate

PO43-, Orthophosphate i TEM Transmission Electron Microscope i

j

IV

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LIST OF FIGURES Figure Page

Figure 1.0 Morphological characters of Pseudo-nitzschia (adopted from Hasle et 4aI., 1996).

Figure 3.1 Kuching map showing the Samariang and Santubong Sampling Site. 9

Figure 4.1 Pseudo-nitzschia spp. temporal distributions at Santubong estuary 16 from September 2011 - April 2012.

Figure 4.2 Pseudo-nitzschia spp. temporal distributions at Samariang estuary 16 from September 2011 - April 2012.

Figure 4.3 In-situ data of temperature, salinity and pH obtained during sampling 19at Santubong estuary water from September 2011 to April 2012.

Figure 4.4 In-situ data of temperature, salinity and pH obtained during sampling 19at Santubong estuary water from September 2011 to April 2012.

Figure 4.5 Macronutrient (Nitrate, Nitrite, Orthophosphate and Silicate) reading 22 at Santubong from 21September 2011 until 2 April 2012.

Figure 4.6 Macronutrient (Nitrate, Nitrite, Orthophosphate and Silicate) reading 23 at Santubong from 21 September 2011 until 2 April 2012.

Figure 4.7 Average precipitation two days before sampling of Kuching from 24 September 2011 to April 2012.

Figure 4.8 Pseudo-nitzschia brasiliana.(A)TEM. Acid-cleaned valve showing 31 the symmetrical frustules (scale bar = 5 flm); (B) TEM. Broadly rounded apical and clear ending valve (scale bar= 2 ,flm); (C) TEM. Broadly rounded apical and clear other ending valve (scale bar= 2 flm); (D)TEM. Clearly visible valvovopula included second and third band. (Scale bar=0.5flm); (E) TEM. Seperated cingular band (Scale bar = 2flm);(F) TEM. Central part of valve without the present of central interspace, striae and 2 rows of poroids are clearly visible (scale bar= 1 flm); (G) TEM. Observation of poroids at high magnification (scale bar= 200 run).

v

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Figure 4.9 Electron micrographs of Pseudo-nitzschia. (A-C) P. pungens. (A) SEM. Portion part of cleaned valve without the present of central interspace (scale bar = 10 Jlm); (B) TEM. Acid-cleaned valve with symmetrical frustules, fibulae, striae and 2 rows of poroids are visible (scale bar = 5 Jlm). (C) TEM.Observation of two row of poroids (scale bar= 0.2 Jlm). (D-F) P. caciantha. (D) TEM of whole cell. (E) TEM. Central part of valve with the number of interstriae, fibulae and one rows of poroids are clearly visible (scale bar = 2 Jlm). (F) TEM. Observation of poroids fonning sectors at high magnification (scale bar = 0.2 Jlm). (G- I) P. circumpora (G) TEM of whole cell. (H) TEM Portion part of cingular band. (I) TEM. Portion central part of valve with the number of striae, fibulae and one row of poroids are clearly visible (scale bar = 0.5 Jlm).

32

Figure 4.12 Maximum likelihood (ML) most parsimonious tree inferred from the LSU ribosomal RNA gene (01-03) sequences of Pseudo- nitzschia species. Tree length was 266 steps, consistency index (CI) is 0.6880 and homoplasy index (HI) is 0.312. Bacillaria paxillifer (Tenerife7), Cylindrotheca closterium (K-520) were the outgroups. Scale bar represents 1 evolutionary step.

35

Figure 4.13 Sequence logos of signature regions for P. circumpora predicted in this study. (A) L-S-Cinn-70-A-21, (B) L-S-Cinn-62-A-21, (C) L-S­Cinn-53-A-71.

39

Figure 4.14 Sequence logos of signature regions for Pseudo-nitzschia caciantha predicted in this study (A)L-S-P-cacian-63-A-19&(B) L-S-P-cacian­63-A-25.

42

Figure 4.15 Sequence logos of signature regions for Pseudo-nitzschia pungens predicte~ in this study.(A) L-S-Ppun400-A-18, (B) L-S-Ppun399-A­18, and (C) L-S-Ppun405-A-18.

45

Figure 4.16 Whole-cell FISH micrographs. (A) P. pungens cells after hybridizing with uniC probe, (B) P. pungens cells after hybridizing with species­specific DNA probe, L-S-Ppun-405-A-18, (C) P. pungens cells after hybridization with UniR, (D) P. brasiliana cells after hybridization with L-S-Ppun-405-A-18.(E)Spiked samples after hybridization with L-S-Ppun-405-A-18.Cells with red fluorescence indicating the presence of chloroplasts.

50

VI

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LIST OF TABLES

Table Page

2.0 Fatality cases due to Domoic Acid production by Pseudo-nitzschia specIes. 6

3.4 Composition of 1 Liter SWII Medium Preparation. 11

4.1

4.2

The clonal cultures maintained in this study provided with strains and locality. Proposed oligonucleotide probe of Pseudo-nitzschia circumpora with probe name, position, signature region, probe sequence, melting temperature (Tm), GC content, Gibb's free energy (~Go ), and hybridization efficiency (HE).

29

40

4.3 Proposed oligonucleotide probe of Pseudo-nitzschia caciantha with probe name, position, signature region, probe sequence, melting temperature (Tm), GC content, Gibb's free energy (~Go ), and hybridization efficiency (HE).

43

4.4 Proposed oligonucleotide probe of Pseudo-nitzschia pungens with probe name, position, signature region, probe sequence, melting temperature (Tm), GC content, Gibb's free energy (~Go ), and hybridization efficiency (HE).

46

Vll

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Ecology and rapid detection of diatom Pseudo-nitzschia (Bacillariaphyceae) at Kuching estuaries

Wang Chuan Yu

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Phytoplankton plays an important role in aquatic ecosystem and serves as sources of foods to support high trophic level in food chains. However, some of the phytoplankton is capable of producing bioactive compound which is harmful to other aquatic organisms. Several species in the genus of Pseudo-nitzschia have been known to produce neurotoxin, domoic acid (DA). In this study, field sampling was conducted fortnightly started from September 20 II to April 2012 to study distribution of genus Pseudo-nitzschia in Kuching estuary water. Abundance of cells was low throughout the study period with exception of April. Pseudo-nitzschia cell isolated from Samariang was successful growth in culture tube and was identified as P.brasi/iana after observed through TEM. The molecular approach Whole-cell fluorescence in situ hybridization (FISH) was developed to detect Pseudo-nitzschia species. The in siJ/ico oligo nucleotide probe was designed for P. pungens (L-S-Ppun-405-A-18), P. circumpora (L-S-P-Cirm-62-A-18), and P. cacianta (L-S-P-cacian-63-A-25) based on sequences obtain in this study and sequences retrieved from SILVA data base. The designed L-S-Ppun-405-A-18 for P.pungens was synthesized with FITC labeled and optimization was carried out for this particular probe. The L-S-Ppun-405-A-18 probe was applied on cultured and spiked samples with UniC and UniR probe as control. The probe showed high specificity toward P. pungens. This study showed FISH is a more reliable in the rapid detection Pseudo-nitzschia cells in HABs monitoring.

Key words: Pseudo-nitzschia; domoic acid; Amnesic Shellfish Poisoning; fluorescence in situ hybridizations (FISH)

ABSTRAK

Filop/anklon memainkan peranan penting da/am ekosistem akuatik dan membeka/kan sumber makanan kepada aras trofik da/am rantaian makanan. Wa/aubagaimanapun, sesetengah fitop/ankton mempunyai keupayaan untuk menghasilkan sebatian bioaktif yang akan membawa mudarat kepada akuatik organism. Beberapa spesies da/am genus Pseudo-nitzschia te/ah dikena/pasti menghasilkan neurotoksin, asids domoic (DA). Da/am kajian ini, pensampe/an te/ah dija/ankan da/am stiap dua minggu bermu/a pada September 20ll hingga April 20 I 2 di muara sungai Kuching untuk menentukan penaburan Pseudo-nitzschia se/. Kepadalan sel ada/ah kurang da/am tempoh kajian kecuali bulan April. Pseudo-nitzschia se/ yang dipenci/kan dari sample Samariang dan berjaya didirikan da/am ku/tur klona/. Se/ dikena/pasti sebagai P. brasiliana se/epas pencirian di bawah Mikroskop Transmisi E/ektron (TEM). Kaedah mo/eku/ se/uruh se/ hibrisasi in situ pendaran (FISH) te/ah dikembangkan dan dipakai untuk mengesankan Pseudo-nitzschia spesies. Oligonuk/eolide probe te/ah direka secara in silico untuk P. pungens (L-S-Ppun-405-A-I8), P. cacianlha (L-S-P-cacian-63-A-25) , dan P. circumpora (L-S-P-Cirm-62-A- I 8) berdasarkan jujukan gen yang dipero/ehi do/am penyelidikan dan pangka/an data SILVA. L-S-Ppun-405A-I8, bagi P.pungens disynlhesiskan dengan FITC label dan pengoptimunan te/ah dija/ankan untuk probe tersebut. L-S-Ppun-405­A-I8 le/ah diap/ikasikan dengan sample ku/tur dan sample kajian dengan UniC dan UniR probe sebagai Jcawa/an. Keputusan ini menunjukkan probe tersebut bertindak secara khusus terhadap P.pungens. Kajian ini menunjukkan kaedah FISH /ebih berkesan da/am pengesanan secara pesat dan pemantau/an HABs.

Kata kunci: Pseudo~nitzschia; Asids domoic (DA); Kerang keracunan amnesic (ASP); hibridisasi in situ pendaran (FISH)

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1.0 INTRODUCTION

The genus Pseudo-nitzschia has more than 30 species and 11 of them had been reported as

potential Domoic acid producers (Lundholm, 2011). According to Wright et a1. (1989),

domoic acid was naturally water-soluble heat stable amino acid. The first report of ASP

(Amnesic Shellfish Poisoning) in Princes Edward Island, Canada, 1987 had confirmed to

be associated with high density of P. multiseries (Bates et aI., 1989) . The ASP event in

Canada had caused three deaths and 105 cases of acute human intoxication after

consumption of contaminated blue mussels(Mytilus edulis) (Bates et a1.,1989). The

consumption of shellfish with highly contaminated of DA would experiences

gastrointestinal distress including vomiting, cramps, diarrhea and short term memory loss

(Perl et aI., 1990).

In Malaysia, there was not any outbreak of ASP case being reported. However,

study of genus Pseudo-nitzschia, had confirmed 6 species existed in Malaysian water

includes P. brasiliana, P. circimpora, P. cuspidata, P. do10rosa, P. micropora, and P.

pun gens (Lim et aI., In press). Based on previous study, occurrence of Pseudo-nitzschia

was shown happened in kuching estuary water (Lim et aI., In press).

The genus of Pseudo-nitzschia can be identified to generic level under light

microscope based on their pennate shape and characteristic chain formation of overlapping

cell tips (Miller & Sholin, 2000). However, species of Pseudo-nitzschia can only be

distinguished by transmission electron microscope which require taxonomic expertise and

trained personnel. The discrimination between different Pseudo-nitzschia species in field

samples is often challenging as it relies on traditional identification method. With the

recent advancement in technology developments, molecular approach such as Whole-cell

Pluorescence in-situ Hybridization (FISH) has been -widely applied in monitoring of

harmful algae blooms (Scholin et aI., 1996). Whole-cell FISH technique is designation of

1

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species specific oligonucleotide probe based on ribosomal RNA sequence in an effort to

speed and quantify the microalgae (Scholin et aI., 1996; Miller and scholin, 1998).

In this study, water samples were collected from Santubong Jetty and Samariang

Batu. The main objective was to study ecology and biology of Pseudo-nitzschia III

Kuching estuary water. The specific objectives of the study as below:

1. To identify the cultured samples of Pseudo-nitzschia species by Transmission

Electron Microscopy (TEM).

2. To detennine the distribution of genus Pseudo-nitzschia in Kuching estuary water.

3. To design in-silica species-specific oligonucleotide probe based on LSU ribosomal

RNA sequence for particular Pseudo-nitzschia species.

4. To Optimize the synthesize probe in hybridization for FISH in detection of Pseudo­

nitzschia species.

2 ....

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1.0 LITERATURE REVIEW

1.1 Harmful Algae Bloom (HABs) Algae bloom is defined as rapid grow and high abundance of single species of algae in

aquatic ecosystem when environmental conditions are favorable. HABs are known as "red

tides" in marine water due to their ability to change color of the water to become red and

results in deleterious effects. Algae bloom can be divided into non-toxic and toxic form.

Hannful algae bloom may cause serious prob ~em in water body and toxic effects depend

on high or low density of toxic algae. However, highly potent harmful algae can cause fatal

to aquatic organisms, human health, and degradation of water quality even in low cell

density (Anderson et aI., 2002). There are many phytoplankton are toxin producers such as

dinoflagellates, cyanobacteria, nuisance macroalgae, and some diatom such as Pseudo­

nitzschia. The genus Pseudo-nitzschia always correlated with aquatic filter feeding

bivalves (Bates et aI., 1989). Bivalves filter feed on high amount of algae regardless of the

toxicity; indirectly neurotoxin would be transferred to higher trophic level through food

web.

1.1 The genus Pseudo-nitzchia

Pseudo-nitzschia is a pennate diatom and some of them are potential DA producers. The

genus of Pseudo-nitzschia was first described by Peragallo & Peragallo (1990) from the

existence genus Nitzchia based on frustule morphology. According to Hasle (1994), the

mmphology of genus Pseudo-nitzchia was first described as colony formed stepped cells,

overlap at the ends, weakly silicified, shallow and flattened valves; eccentric raphe, not

elevated above general level of valve, lack of conopea, and striated girdle band

Cbaracterize.

3

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According to Lundholm (2011), eleven specIes of genus Pseudo-nitzachia have

been reported as potential producers of DA toxins, i.e. P. pungens, P. australis, P.

calliantha, P. cuspidata, P. delicatissima, P. fraudulenta, P. multiseries, P. multistriata, P.

seriata, P. pseudodelicatissima, and P. turgidula.

Scanning Electron Microscope and Transmission Electron Microscope are widely

used to identify Pseudo-nitzchia until species level. The species of Pseudo-nitzschia can be

classified through morphological characters such as, width of valve, shape of valve,

density of interstriae and fibulae, central interspace, structures of fibulae and structures of

striae (number of rows, density of poroids) (Figure 1.0).

,n,...wa-I stria(e) '= '""

overlap ­of

callands central interspace with central nodule and raphe endings

valve girdle view view

Figure 1.0: Morphological characters of Pseudo-nitzschia (adopted from Hasle et al. 1996)

4

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Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWA)(

1.3 Amnesic Shell Fish Poisoning (ASP)

ASP is a type of intoxication caused by some toxic Pseudo-nitzschia. DA was found

n:sponsible for ASP (Bates, 1998). The bloom of DA producing Pseudo-nitzschia sp. have

been responsibility numerous mortality of birds and marine mammals in California

(Anderson et aI., 2006). According to Bargu et at. (2002), zooplankton grazer community

is a key of potential vector in transferring DA to high trophic level. The first reported of

ASP in Canada 1987 has attracted attention of public on seriousness of the neurotoxin.

Human infected ASP through consumption of contaminated shellfish that have

accumulated high DA. ASP also has a significant effect on marine mammals and multiple

poisoning events over the last decades (Table 1.0).

DA is a heat stable and water-soluble tricarboxylic amino acid that acts as an

8D8log of the neurotransmitter glutamate (Wright et aI., 1989). DA has high potent bind at

1cainite, a-amino-5methyl-3hyroxyisoxazolone-propionate and N-methyl-D-aspartate

subclasses of inotropic glutamate receptors (Lefebvre & Robertson, 2010). The interaction

of exogenous DA with 3 subclasses of inotropic glutamate receptors will cause influx of

cessive Ca+ at synaptic neuron which might disrupt cellular function. The consumption

of shellfish contaminated with DA would experiences gastrointestinal distress including

vomiting, cramps, and diarrhea (Perl et aI., 1988). In addition, patient ingested DA with

<CODCClDtration more than 1.9mg/kg body weight will cause neuron cells death at the region

hippocampus due to disruption of Ca+ dependent cascade effect (Perl et aI., 1990;

Lefebvre & Robertson, 2010).

5

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Table 1.0: Fatality cases due to Domoic Acid production by Pseudo-nitzschia species.

Year location Pseudo-nitzschia vector Consequences References S(!ecies Im(!licated.

1987 Canada, P. multi series Blue mussel 153 cases of acute Bates et aI., 1989 princes intoxication and 3death Edward island

1991 California P. australis Anchovies Pelicans and Work et aI., 1993 cormorants Poisoned

1994 Oregon and Razor clam 25 illness Bates et aI., 1998 Washington and

Dungeness crab

1998 Monterey bay P. australis and P. Anchovies 70 female sea lions Scholin et aI. , California multiseries and sardines died 2000

2000 Turkish water P. australis Krill Bargu et aI. , 2002

2003 Santa Barbara P. australis Marine mammals Anderson et aI., Channel, mortality 2006 California

2.4 Factors influence the growth of Pseudo-nitzschia

2.4.1 Environmental Factors

Abiotic factors are defined as non-living chemical and physical parameters in environment.

All aquactic biological changes are affected by environmental factors . According to

Fehling et al.(2004), increase in cell density of difference Pseudo-nitzschia sp. could be

explained by variable environment parameters such as temperature, light and nutrients.

2.4.2 Temperature and Irradiance

Water temperature is defined as measurement of hotness or coldness of water. The

fluctuation of water temperature may be due to the season, time of day and weather. Light

intensity is related to the solar radiation which might differed in difference seasons and

water transparency. The illumination of sunlight will give rises to water temperature. Light

and temperature would detennine the biogeographical distribution of some phytoplankton

species. According to Lewis et al.( 1993), P.pungens culture show maximum yields of cell

stationary phase in low temperature conditions.

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2.4.3 Salinity

Salinity is a measurement of dissolved ions in water expressed in PSU. Majority of

Pseudo-nitzschia sp. are euryhaline and physiological adapted to changes of salinities

(Bates et aI., 1998). Pseudo-nitzschia sp. was also reported to grow well in salinity up to

45Psu (Thessen et aI., 2005).

2.4.5 Nutrients

Seawater is rich of inorganic nutrients. The major composition of inorganic nutrients in

seawater are inorganic nitrogen compund (N02+,N03+,NH/), phosphate and silicate.

Inorganic nutrients in water body play an important role in influencing the growth and DA

production ofPseudo-nitzschia sp. (Trainer et aI., 1998; Wells et aI., 2005; Schnetzer et aI.,

2007,). According to Trainer et a1. (1998), cells that receive nitrate as nutrients triggering

the intial Pseudo-nitzschia bloom in Pen Cave, while limitation of iron, silica, and

phosphate has created physiological stress to Pseudo-nitzschia in acceleration of DA

production (Pan et al. 1996; Schnetzer et a1. 2007; Well et aI., 2005 ).

Whole- cell Fluorescence in-situ Hybridization (FISH)

has been shown to be a suitable tool for determinative phylogenetic and

sequence of single strand DNA that complementary to the DNA sequences of the

organism. Whole-cell FISH with designed rRNA-targeted oligonucleotide probes

bind to whole cell of target organism. According to Lipski et al. (2001), ribosomal

an excellent targeted molecule due to high natural concentration and high

._Iti(]ln content to provide signature nucleotide content for most phylogenetic taxa. . .

lication of whole-cell FISH had shown applicable in identifies and enumerates

7

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ful Pseudo-nitzschia sp. in culture sample and environmental sample (Miller & Sholin,

20(0). The running of Whole-cell FISH protocol involves several steps : fixation of cells,

mounting and observation under fluorescence

·croscope (Miller & Sholin, 2000).

8

,'­

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3.0

3.1

Plankton samplings

MATERIALS AND METHODS

Sampling Site

were carried out at Santubong Jetty (1.7164 N, 110.3281 E) and

Samariang Batu (1.6092 N, 108 110.3244 E). Santubong Jetty was located at the river

mouth of Santubong River, meanwhile Samariang Batu was located near the Shrimp

Aquaculture sites. Field samplings were undertaken fortnightly during high tide from

September 2011 to April 2012.

Kuching

Figure 3.1: Kuching map showing the Samariang and Santubong Sampling Site

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.2 In situ Measurement

Samples were collected by using 20Jlm plankton net with vertical haul for qualitative

aualysis. Van dom water sampler was used to collect samples for quantitative analysis.

One liter plankton samples were collected in duplicates at each site. All samples were kept

in cooler box and brought back to the laboratory for further analysis. In situ parameters of

salinity, temperature, and pH were measured .

.3 Cell Counting

Samples were concentrated through filtration. Duplicate 20 mL concentrated samples were

preserved one with acidic Lugol solution and another with saline ethanol (Scholin et aI.,

1996). The filtrates were used for nutrients analyses.

Cell counting was conducted on the Lugol preserved samples. Total of 1 mL

subaample was pippetted onto a Sedgwick-rafter counting chamber, and the Pseudo-

IiItzIchia cell abundances were enumerated under a light microscope. Cell count was

t*fonned in triplicates. Cell enumeration data from the field samples was recorded and

.uJated, with total cell density (D) calculated based on formula as below:

n D (cells C 1

) = CF x 1000

Original sampling volume (mL)

Final concentrated volume (mL)

Cell Isolation

.~l.8IIlPlc~ were used for clonal culture establishments. Samples were placed in a petri

observed under an inverted light microscope. The target cell was isolated through

.,.petling technique. The fine capilary Pasteur pipett-e was carefully dipped into the

and Pseudo-nitzschia cell was drawn into the pipette through capillary action. A

10

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single chain cell was transferred into a drop of filtered seawater to wash away debris and

unwanted cells. The transferring process was repeated until a clean single chain cell

obtained. The cell was inoculated into a 96-well microplate containing filtered seawater

and maintained in the light temperature controlled incubator.

3.5 Medium Preparation

For culture maintenance, SWII culture medium was prepared according to Iwasaki (1961).

atural seawater was used as medium base. The SWII medium was prepared by adding

03, KH2P04, Na2-glycero.P04, Fe-EDTA, Tris-HCI, (Vitamin mix (mixture of B12

cyanocobalamin), biotin, and Thiamine-HCI), and silicate (Table 3.1). The pH of medium

adjusted to 7.8- 7.9 by using 10% HC!.

able 3.1: Composition of SWII Medium (Iwasaki 1961)

Stock Concentration Volume Final Concentration (moll L) (mL) (moll L)

7.2x 1 1.0 7.2x 1 3.31 x 10-2 1.0 3.31 x 10-5

3.33x 1 0-2 1.0 3.33xlO-5

1.0 1.19xl0-6

iftiI..HCl(pH 7.8) 5.0 4.13x 10-3

R_iD Mix B12(cyanocobalamin) 4.43x 10-10

o biotin 4.1 x 10-9

Thiamine-HCI 3.0x 10-7

0.5

When cell division was observed in the well, the cells were transferred to a test tube

25mL SWII medium. The transferring process was carried out aseptically in a

flow to avoid contamination. Cultures were maintained at 25°C, under light

0(70 J.lIllole photon m_2·s) with 12:12 h light: dark cycle.

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.6 Acid Wash of Pseudo-nitzchia Samples and TEM Observation

Pseudo-nitzchia cells were observed under a Transmission Electron Microscope (TEM).

SImples were treated with acid wash (Hasle & Fryxell, 1970) before observing under TEM.

tal of 20 mL samples were transferred into 50 mL centrifuge tube and equal volume of

98% H2S04 was added. Saturated KMn04 was added to the centrifuge tube until mixture

tum purple, immediately 10% Oxalic acid was added until mixture turned clear to remove

04. Samples were centrifuged at 8000 rpm for 10 min at room temperature.

upematant was discarded and distilled water added to rinse the samples. The rinsing

p.ocess was done several times to remove salt and acid.

Acid-wash samples were then transferred and mounted onto a Fomvar coated 300

copper grid. Cells were then observed under a JiSM-1230 TEM (lEOL, Japan) and

was identified until species level based on detailed morphological

Nutrients Analysis

_red water sample was used for nutrients analysis. Total of five treatments (Blank

pol, N03-, Si02 and N02-) were prepared by rinsing the cells with water sample

adding 10 mL of water samples. The reagents were added into the cells and let the

take place at different time according to the Hach Manual Dr 12010 (Hach, USA).

blank cell was placed inside HACH DR2800 for zeroing before readings of water

The inorganic nutrients were analyzed in triplicates.

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3.8 Phylogenetic analyses

cleotide sequences of large subunit (LSU) ribosomal rONA gene of Pseudo-nitzschia

species and two outgroups (Fragilaria capucina and Bacillaria paxill~fer) were retrieved

tom Genbank nucleotide database (NCBI) (AppendixE). Four sequences of LSU rDNA of

P. eircumpora and P. caciantha were obtained from Unimas Harmful Algal Group. All

U rDNA sequences were initially aligned using ClustalX 2.0 (Thompson et al., 1997)

tnd further edited manually by BioEdit ver. 7.0 (Hall, 1999). The output of the multiple

aligoments was saved in Clustal, Fasta and Nexus format. The phylogenetic inference,

tiple-alignment of LSU rDNA sequences obtained were used to reconstruct the

fogeny using PAUP* ver. 4.0 (Swofford, 2000). The phylogenetic tree were

-Cltft'cted in three program which were included Maximum Parsimony (MP), Maximum

rftIdlKx:K1 (ML), and Bayesien inference (BI) to increase the accuracy of branch support.

In silieD oligonucleotide probe design

3IJ1QO-nll'ZScma species LSU rDNA sequences were retrieved from SILVA database, and

saved as ARB format (Appendix E). The ARB package (Ludwig et aI., 1994) was

analyze the signature sequences of LSU rDNA and search PT server for potential

·tcs. The ARB 'Probes Match' program was used to examine the proposed

.~ott(le probes. At least one mismatch was observed between different species.

parameters such as probe length, melting temperature, GC content, Gibbs' free

E-value were obtained using the Web interface, Intergrated DNA

_pes (IOn. Basic Local Aligment Search Tool (BLAST) was used for further

I!.I!'I'!"'taVN and specificity test of the probes.

13