MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF RIBOSOMAL DNA
SEQUENCES OF A HARMFUL ALGAL BLOOM SPECIES, PYRODINIUM BAHAMENSE
VAR. COMPRESSUM
Chin W.L., Teoh P.L., Anton A. and Kumar S.V.
Biotechnology Research Institute, University Malaysia Sabah,
Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia.
Email: [email protected]
ABSTRACT
Harmful algae blooms in Sabah only occur in the coastal waters of west Sabah, where one
of the causative organisms is the marine dinoflagellate, Pyrodinium bahamense var.
compressum. For fast and accurate identification of this harmful alga, study has been carried
out to develop a DNA probe from highly conserved small-subunits ribosomal DNA region.
Five seawater samples containing P.bahamense, G1, KBE3, PG, PS and PYRO, were
isolated and cultured in f/2 medium. After DNA extraction, PCR amplification was carried out
targeting the rDNA of the algae. The size of the PCR product yielded was approximately 1.6
kb. A total of eight restriction enzymes, HaeIII, DpnII, AluI, RsaI, BsaJI, BstNI, BstUI and
TaqI, were used to determine the homology with the five samples. The results of the
restriction enzyme digestion indicated that all five P. bahamense samples were similar in
their restriction enzyme patterns. Besides that, sequences of all the five samples were
successfully obtained. The sizes of sequences for sample G1, KBE3, PG, PS and PYRO
were 1,545 bp, 1,580 bp, 1,564 bp, 1,544 bp and 1,576 bp respectively. The sequences of
samples G1, KBE3, PG, PS and PYRO were compared to the sequence of 18S SSU rDNA
of Pyrodinium bahamense from GenBank, there was 3, 1, 3, 2 and 4 nucleotides differences
respectively. Once the most conserved region of Pyrodinium bahamense var. compressum
have been identified, it will then serve as a unique identifier for this species.
INTRODUCTION
Harmful algae are planktonic algae or phytoplankton. These algae serve as energy provider
at the base of the marine food web for filter-feeding bivalve shellfish, such as oysters and
mussels. Occasionally, the algae multiply or ‘bloom’ until they reach such high
concentrations (up to millions of cells per litre) that the surface of the sea becomes
discolored, often called the red-tides. Harmful algal bloom (HAB) is usually not harmful, but
small percentages of the blooms are considered harmful because the algae can produce
potent natural poisons known as biotoxins (Rhodes, 1998). Proliferation of these algae
causes massive fish kills, contaminates seafood with toxins and also alters the ecosystems
in ways that eventually threaten the human life. Harmful algal blooms in Sabah only occur in
the South China Sea in the coastal waters of the west Sabah. It was first reported since
1976 (Azanza & Taylor, 2001), where one of the causative organism is the dinoflagellate,
Pyrodinium bahamense var. compressum (Anton et al., 2000).
The species, Pyrodinium bahamense var. compressum is an armored, bioluminescent
dinoflagellate, and they are the major species involved in the tropical Indo-Pacific red tides.
(Badylak et. al., 2004). This HAB species produces saxitoxin, as a by-product, in filterfeeding bivalve shellfish that can cause Paralytic Shellfish Poisoning (PSP) that eventually
causes paralyses and deaths in humans. Saxitoxins are neurotoxins that act to block
movement of sodium through nerve cell membranes, stopping the flow of nerve impulses
causing the symptoms of PSP, which then causes symptoms such as, numbness, paralysis,
disorientation and also death through repiratory paralysis. There is no antidote for PSP.
Malaysia has reported a total of 609 PSP cases and 44 deaths (Azanza and Taylor, 2001).
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Identification of HAB species in discrete water samples has relied traditionally on
microscopy. Although, the traditional microscope-based method has been proven reliable,
this method is time-consuming and requires expertise to recognize the different
morphological characters of the HAB species (Scholin and Anderson, 1998). Besides that,
some species exists as a variety of strains, or ‘subspecies’, that are not possible to delineate
using morphological criteria. Therefore, molecular studies of HAB will be very beneficial in
species identification as the morphological features of these species may change or evolve
with environmental conditions or life history.
Molecular or DNA probes assays provide fast and accurate descriptions of different kinds of
HAB species. DNA probes are probes that detect specific DNA sequences that serve as
unique identifiers of a species or group of species (Scholin & Anderson, 1998). Besides that,
they also speed up and refine analyses of seawater samples for faster or rapid detection and
identification of potential toxic blooms. The nucleotide sequence of a selected micro-alga
that makes up its genetic code is compared with that of other related algae. Unique
segments (consists of 15 to 50 nucleotides) are selected to develop into probes. In this
research, the main focus is on designing a DNA probe that is specific for the identification of
HAB species, Pyrodinium bahamense var. compressum. The ribosomal DNA (rDNA) of the
cell is targeted for recognition. In this study, comparison of DNA fingerprints of P.bahamense
collected from various locations was also done based on their 18S rDNA sequences.
OBJECTIVES

To develop a DNA probe from highly conserved small subunits rDNA region for
Pyrodinium bahamense var. compressum, one of the causative HAB species in Sabah.

To compare the DNA fingerprints of plankton cell of Pyrodinium bahamense var.
compressum samples collected from the west coast of Sabah based on their 18S SSU
ribosomal DNA sequences.
MATERIALS AND METHODS
Study Area
Sampling of P.bahamense was carried out in the coastal waters of west Sabah, where HAB
species was sampled particularly in areas where they have been previously reported or
identified.
Collection and Isolation of Algae
At each sampling site, triplicate seawater samples were collected for laboratory analysis.
Plankton net with the mesh size of 20 μm was used to scoop the seawater from the
dockside, shore or alongside the boat and transferred in plastic bottles. Freshly collected
seawater samples were examined under a light microscope and single-cells of P.bahamense
were isolated using drawn-out micropipettes. P.bahamense cells were washed using drops
of water on slides to make sure minimum contaminations occurred.
Algal Culturing
P.bahamense algae were then cultured in f/2 medium (Guillard and Ryther, 1962; Guillard,
1975). All cultures were maintained at 25 to 28 °C with a photoperiod comprising 14 hours of
light and 10 hours of darkness and also under the light intensity of 3,800 to 4,500 lux.
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DNA Extraction
After the cultures had reached their mid-exponential growth rate, the P. bahamense cells
were harvested by repeated centrifugation until the desired volume was obtained. DNA was
extracted using a modified CTAB (cetyltrimethylammonium bromide) DNA extraction
reported by Doyle and Doyle (1987). The cells were resuspended in CTAB buffer containing
100 mM Tris (pH 8.0), 1.4 M NaCl, 20 mM ethylenediamine tetra-acetic acid (EDTA), pH 8.0,
and 2 % CTAB, and incubated at 65 °C for one hour. The mixture was extracted twice with
equal volumes of chloroform-isoamyl alcohol (24:1). DNA was precipitated by the addition of
⅔ volume of 100 % ice-cold isopropanol. The DNA was rinsed with 70 % ethanol and
dissolved in TE buffer (10 mM Tris, pH 8.0; 0.1 mM EDTA, pH 8.0). The DNA samples were
then kept at –20 °C. Before PCR, gel electrophoresis on a 1% agarose gel was done to
determine the quality of DNA samples.
Polymerase Chain Reaction Amplification
The polymerase chain reaction (PCR) primers used in this study were TPL1F (5’TAAGCCATGCATGTCTCAG-3’) and TPL1R (5’-GGATCACTCAATCGGTAGG-3’). Both
primers were designed based on P.bahamense small subunit rRNA gene sequence in the
GenBank database (AF274275) with the size of 1,754 bp.
PCR was carried out in a 50 μl reaction mixture containing 2.5 U ProofStart DNA
polymerase, 1 x Proofstart PCR buffer, 1 x Q-solution (Qiagen USA), 300 μM dNTPs
(Promega, USA), 1 μM each primer and 100 ng – 1 μg genomic DNA. PCR amplification
was performed as follows: 5 minutes initial activation step at 95 °C, followed by 35 cycles of
denaturation at 94 °C for 1 minute, annealing at 55 °C for 1 minute, and extension at 72 °C
for 2 minutes, and the final extension at 72 °C for 7 minutes. The PCR was carried out on a
PTC-200 thermal cycler (MJ Research, USA). The PCR products were then analyzed on
2% agarose gel. The desired bands were excised from the agarose gel and purified by using
the QIAquick Gel Extraction Kit (Qiagen, USA) according to the manufacturer’s instructions.
The purified products were then subjected to direct sequencing and restriction enzyme
digestion.
Restriction Enzyme Analysis
A total of 5 μl of purified PCR product was digested with eight restriction endonucleases,
HaeIII (Promega Corp.), DpnII, AluI, RsaI, BsaJI, BstNI, BstUI and TaqI (New England
Biolabs, USA). Restriction enzyme analysis was carried out in a 10 µl reaction mixture
containing 1 x restriction enzyme buffer, 10 µg/µl BSA and 5.0 µl (1:2 v/v) of DNA template.
Restriction fragments were then analyzed on a 1.5 % agarose gel.
Multiple Sequence Alignment
The sequences of the five samples of P. bahamense were assembled using SeqManII
version 6.1 software (DNASTAR, Inc., USA). The sequences of the sampless were then
aligned using CLUSTAL W version 1.81 software. The sequences were compared to each
other and also compared with the 18S rDNA sequences of P.bahamense in GenBank
(AF274275).
Primer Design
P. bahamense sequences will be aligned and compared with other dinoflagellate sequences
available in the Genbank database, where the conserved region of P.bahamense will be
identified.
Once the conserved region has been identified, a specific primer for
P.bahamense will then be synthesized by using Primer3 software and will be tested against
selected dinoflagellate species available in the laboratory.
Development of DNA Probe for Pyrodinium bahamense var. compressum
Once the conserved region of P.bahamense var. compressum species have been identified
and confirmed, DNA probe labelled with fluorescent will be developed. The specification of
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this probe will then be verified by population testing on the pure cultures of the selected
species obtained from the culture collections and also field-samples.
RESULTS AND DISCUSSION
Algal Isolation and Nucleic Acid Extraction
A total of five P.bahamense samples, G1, KBE3, PG, PS and PYRO, from four different
offshore locations had been isolated and established in culture. The genomic DNA from all
the five samples was successfully extracted using the CTAB DNA extraction method (Figure
1). All four samples extracted were of highly compact bands, with faint smear below the
bands. The quantity and quality of the DNA extracted was of PCR grade.
bp
M
1
2
3
4
5
23,130
Figure 1 DNA samples extracted using CTAB DNA extraction method. Lane
M: Lambda DNA/HindIII Marker; Lane 1: G1; Lane 2: KBE3; Lane 3: PG;
Lane 4: PS; Lane 5: PYRO.
PCR Amplification
All DNA samples extracted by the CTAB extraction method were subjected to PCR
amplification. PCR amplification of the five samples resulted in a single highly compact band
with two faint bands below (Figure 2A). The highly compact band of the five samples was
excised from the agarose gel and was then purified using QIAquick Gel Extraction Kit
(Qiagen, USA). This resulted in a single product of approximately 1,600 bp (Figure 2B).
bp
1,500
M1
1
2
3
4
5
6
M2
bp
1,500
bp
1,500
Figure 2A PCR products of P.bahamense samples. Lane
M1
1
2
3
4
5
M2
bp
1,500
Figure 2B Purified PCR products of P.bahamense samples.
Lane M1: 100bp DNA ladder; Lane 1: G1; Lane 2: KBE3;
M1: 100bp DNA ladder; Lane 1: G1; Lane 2: KBE3; Lane
3: PG; Lane 4: PS; Lane 5: PYRO; Lane 6: negative
control; Lane M2: 1 kb DNA ladder.
Lane 3: PG; Lane 4: PS; Lane 5: PYRO; Lane M2: 1 kb
DNA ladder.
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Restriction Enzyme Analysis of Pyrodinium bahamense var. compressum
All eight restriction enzymes, HaeIII, DpnII, AluI, RsaI, BsaJI, BstNI, BstUI and TaqI, yielded
similar restriction enzyme (RE) pattern among the five P.bahamense samples (Figures 3A,
B, C, D, E, F, G and H). The recognition sites of all eight REs are listed in Table 1. The RE
analysis suggests that all five P.bahamense samples were derived from the same variety. In
addition, a virtual restriction mapping of 18S rDNA sequence of P. bahamense for the eight
restriction enzymes was also conducted using RestrictionMapper version 3 (Brakmann,
2002). The results of the virtual mapping were similar with the RFLP analysis results. These
results suggest that all five samples were from the same homogenous population. The RE
analysis reveals that all five P.bahamense samples probably representing homologous
stretches of DNA (Dowling et al., 1996). Nuclear rDNAs analysis has been chosen to be the
technique of choice for molecular ecological and evolutionary studies of extant
dinoflagellates, because it had been reported that rDNA coding regions exhibit significantly
higher sequence conservation (Bhattacharya et. al. 1991: Santos et. al., 2003).
Table 1 Recognition site of each restriction enzyme used in this study.
Restriction Enzymes
HaeIII
DpnII
AluI
RsaI
BsaJI
BstNI
BstUI
TaqI
Recognition Site
GGCC
GATC
AGCT
GTAC
CCNNGG
CCWGG
CGCG
TCGT
Overhang
Blunt
5’
Blunt
Blunt
5’
5’
Blunt
5’
DNA Sequencing and Data analysis
Purified PCR products of all samples subjected to direct sequencing. The primers used for
sequencing reactions are shown in Table 2. Sample G1 and PS only required 2 primers,
TPL1F and TPL1R, to obtain the full sequence, whereas for sample KBE3, PG and PS,
three partial sequences were generated using the two mentioned primers and an additional
primer, P1 (5’-CAGCCTTGCGACCATACTC-3’).
Table 2 Primers used in sequencing reactions of the five
Samples
G1
KBE3
PG
PS
PYRO
Forward Sequencing
Primer(s)
TPL1F
TPL1F
TPL1F
TPL1F
TPL1F
Reverse Sequencing
Primer(s)
TPL1R
TPL1R, P1
TPL1R, P1
TPL1R
TPL1R, P1
All five sequences were assembled using SeqManII version 6.1 software (DNASTAR, Inc.,
USA). The sizes of sequences for sample G1, KBE3, PG, PS and PYRO were 1,545 bp,
1,580 bp, 1,564 bp, 1,544 bp and 1,576 bp respectively. Multiple sequence alignment of the
five samples was done using Biology Workbench version 3.2 Nucleic Tools via CLUSTAL W
version 1.81 software, where P. bahamense from the GenBank (AF274275) were used as a
the backbone reference. Alignment results showed that all five samples’ sequences have
high similarity with only one to four nucleotides differences. All the samples have up to 99%
similarity with the sequence deposited in the Genbank. Number of nucleotide differences
and percentages of similarity of all P.bahamense samples are shown in Table 4. The
nucleotides differences maybe due to presence of artifacts of the PCR amplification and / or
sequencing method employed (Scholin & Anderson, 1996), or maybe it is due to mutation of
the algae for adapting to the new environment.
5
bp M1 1
2
3
4
5
6
7
M2 bp
bp M1
A
1
2
3
4
5
6
7
M2 bp
B
1,500
1,500
1,000
900
600
200
bp M1 1
2
3
4
5
6
7
M2 bp
bp M1
1
2
3
4
5
6
7
M2 bp
D
C
1,500
1,500
1,000
600
500
400
200
bp M1
E
1
2
3
4
5
6
7
M2 bp
F
bp M1
1
2
3
4
5
6
7
1,500
1,500
M2 bp
1,000
500
300
200
G
bp M1
300
200
1
2
3
4
5
6
7
M2 bp
bp M1
1,500
C
1
2
3
4
5
6
7
M2 bp
H
1,500
900
400
500
400
200
200
Figure 3 Digestion of the PCR products with HaeIII (A), DpnII (B), AluI (C), RsaI (D), BsaJI (E), BstNI (F), BstUI
(G) and TaqI (H). Lane M 1: 100 bp ladder marker; lane 1: G1; lane 2: KBE3; lane 3: PG; lane 4: PS; lane 5:
PYRO; lane 6: negative control; lane 7: undigested PCR product; lane M2: 1 kb ladder marker.
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Table 3 Number of nucleotide differences and percentages of similarity in the sequences
generated from all five P.bahamense samples with the sequence of P.bahamense from the
GenBank (AF274275) as the backbone reference.
Samples
G1
KBE3
PG
PS
PYRO
Number of Nucleotide
Differences
3
1
3
2
4
Percentages of
Similarity (%)
99.8
99.9
99.8
99.9
99.7
Besides that, sequence comparison of four samples, G1, PG, PS and PYRO, was also done
with the sequences that were previously obtained by Chan (2004). There were 9, 4, 4 and 5
nucleotides differences respectively. Besides for the mentioned reason above, the
nucleotides differences may also due to the types of DNA polymerase used in both study.
Pfu DNA polymerase was used in this study, whereas in Chan’s study, Taq DNA polymerase
was used. It had been reported that the error rate of Pfu has been found to be ~ 10-fold
lower that that of the non-proofreading enzyme Taq (Cline et. al., 1996).
PROPOSED SCOPE OF WORK
In future, the conserved region of P.bahamense will be identified by comparing its the 18S
rDNA sequences with other dinoflagellates’ 18S rDNA sequences. Once the conserved
region has been identified, a specific primer for P.bahamense will be synthesized by using
primer3 software and will be tested against selected dinoflagellate species available in the
laboratory.
After the conserved region of Pyrodinium bahamense var. compressum species have been
identified and confirmed, DNA probe labeled with fluorescent will be developed. The
specification of this probe will then be verified by population testing on the pure cultures of
the selected species obtained from the culture collections and also field-samples.
ACKNOWLEDGEMENT
The authors thank University Malaysia Sabah for supporting this study by providing the
fundamental grant (B-09-01-13-PR/U018).
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