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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
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)
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
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.
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 ....
,..
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
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.
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VI
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
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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).
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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
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)
V111
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
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 ....
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
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
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
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
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
,'
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
9
.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
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.
11
.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.
12
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