Post on 01-May-2023
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PYRODINIUM BAHAMENSE VAR. COMPRESSUM
TO VARYING SALINITY-TEMPERATURE CONDITIONS
ALICE ILAYA GEDARIA
A Master's Thesis Submitted to the
Institute of Biology
College of Science
University of the Philippines
Diliman, Quezon City
As Partial Fulfillment of the Requirements
for the Degree of
MASTER OF SCIENCE IN MICROBIOLOGY
November 2002
GROWTH RESPONSE AND TOXICITY OF CULTURED
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This is to certify that this master's thesis, entitled "Growth response and
toxicity of cultured Pyrodinium bahamense var. compressum to varying salinity-
temperature conditions" and submitted by ALICE ILAYA GEDARIA to fulfill part
of the requirements for the degree of Master of Science in Microbiology was
successfully defended and approved on November 11, 2002.
ERNELEA P. CAO, Ph.D.
Thesis Adviver
RHODORA V. AZANZA, Ph.D. MILAGROSA MARTINEZ-GOSS, Ph.D.
Thesis Co-Adviser Thesis Reader
The Institute of Biology endorses acceptance of this master's thesis as partial
fulfillment of the requirements for the degree of Master of Science in Microbiology.
NELLIE LOPEZ, Ph.D
Director
Institute of Biology
This master's thesis is hereby officially accepted as partial fulfillment of the
requirements for the degree of Master of Science in Microbiology.
RHODORA V. AZANZA, Ph.D.
Dean, College of Science
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ACKNOWLEDGEMENT
My deepest thanks to the following people who helped me in this study:
The Harmful Algal Bloom Laboratory, Marine Science Institute, University of the
Philippines, Diliman for the facilities.
Dr. Rhodora V. Azanza, for giving me the opportunity to do this study, for all the
assistance and encouragement. Thank you very much.
Dr. Christian Hummert, Bernd Luckas and Catherine Reinhardt from Friedrich-
Schiller University, Germany for analyzing the cell samples in HPLC. I really
appreciate your effort in analyzing my numerous cell samples.
Dr. Ernelea P. Cao, my thesis adviser for all the assistance and kind help to finish my
thesis. Thank you for all the extra effort you’ve exerted in finalyzing my manuscript
and thesis defense.
Dr. Milagrosa Martinez-Goss, my thesis reader for all the useful suggestions to
improve my study.
To Dr. Patricia V. Azanza for the guidance and encouragement to finish my studies.
Thank you so much.
My co-workers, Lilibeth, Alette, Claudette, Iris and Ate Lits for extending their kind
help while doing my experiment. Thanks for all the encouragement, support and
friendship you’ve given me. To Daisy Padayao for the use of spectrophotometer. Ate
Arlene Boro for the computer and LCD Projector.
My best friend, Lilibeth for helping me do the sampling during weekends and for the
useful comments and suggestions. Thank you for standing by me through “thick and
thin”. I will treasure you for the rest of my life.
My family, Mama, Papa, Christopher, Aris and Ruby for the love and understanding.
Momy Elvie, Tita Ness, Tita Anne and Sanse for giving me support to finish my
studies. I will never forget that.
To my inspirations. Jao and Jara. You made me a stronger person. This is for you.
Above all, to “God Almighty” for all the blessings and guidance. Thank you for
leading my life.
Alice Ilaya Gedaria
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ABSTRACT
The growth and toxin production of a Philippine Pyrodinium bahamense isolate in
nutrient replete batch cultures were investigated at various stages of the life cycle and
under conditions affected by varying salinity, temperature and combined effects of
salinity and temperature. The organism was routinely maintained in sterile seawater
enriched with F/2 medium at 24±2°C, 150 uEm-2s-1 under a 12: 12 h light: dark (L:D)
cycle. Early exponential growth stage was reached after 7 days with a cell division
rate of 0.26 div day-1. The toxin content reached a peak of 298 fmol cell-1 at mid
exponential phase (14 days) and rapidly declined to 54 fmol cell-1 as the organism
approached the death phase. Only three sets of toxins composed of STX, dcSTX and
B1 were detected. NeoSTX, GTX1-6 and C toxins were not produced during the
entire growth cycle. The organism was able to grow from 26 to 36 0/00 with an
optimum growth at 36 0/00. Growth rate was highest at 36 0/00 (0.4 div d-1) while the
toxin content was highest at 30 0/00 (260 fmol cell-1). A decline in STX composition
(from 80 to 65 mole %) was observed with increased salinity while dcSTX
composition increased from 15 to 32 mole %. B1 toxins remained constant in all the
salinity variations conducted. P. bahamense was able to grow from 23 to 36°C and
the optimum temperature for growth was at 25°C. Lowest growth rate (0.22 div day-1
) was observed at 25°C, while highest growth rate was achieved at 0.4 div day-1 at
35°C. Toxin content reached a peak of 376 fmol cell-1 at 25°C and was lower (80 to
116 fmol cell-1) at higher temperatures (32 to 35°C). STX made up to 85 to 98 mole
% toxin cell-1. However, a 50% decrease in mole % toxin cell-1 STX was observed at
36°C with increased proportion of dcSTX and B1. No dcSTX was produced at
temperatures from 25 to 34°C. Combined effects of salinity and temperature showed
that P. bahamense was not able to grow at low salinity and temperature (26 0/00 –
28°C). Optimum growth was observed in higher salinities at all temperature
conditions. Highest specific growth rate was 0.4 div day-1 at 30 0/00 - 32°C, while
toxin content peaked at 30°-25 0/00 amounting to 665 fmol cell-1. STX comprised
about 80 to 99 mole % toxin cell-1.
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TABLE OF CONTENTS
TITLE PAGE ………………………………………………….……………….. i
ENDORSEMENT PAGE ……………………………………………………… ii
ANNOUNCEMENT …………………………………….……………….……. iii
ACKNOWLEDGMENT……………………………………….…………….…. iv
ABSTRACT ……...…………………………………….…………….…….….. v
LIST OF FIGURES …………………………………………………………… vii
LIST OF TABLES ……………………………………………………………. viii
I. INTRODUCTION ………………………………….……………………….
1
II. REVIEW OF LITERATURE …………………………………………….
3
III. MATERIALS AND METHODS …………………………………...……
13
A. Growth Curve of Pyrodinium bahamense var. compressum ..….…. 13
B. Salinity Effects ………………………………………………….… 15
C. Temperature Effects …………………………………………….…. 16
D. Combined salinity and temperature effects …………………….….. 17
E. HPLC Analysis …………………………………………………..… 18
IV. RESULTS ………………………………………………………….…..…. 22
V. DISCUSSION ………………………………………………………………
A.Culture of Pyrodinium bahamense var. compressum ..……………... 25
B. Toxin Composition ..………………………………………………. 26
C. Cellular Toxin Dynamics ..………………………………………… 29
D. Salinity Effects ………………………………………………….….. 30
E. Temperature Effects …………………………………………….….. 32
VI. CONCLUSION ……………………………………………………….…... 34
VI. LITERATURE CITED …………………………………………….…….. 35
VII. LIST OF FIGURES ……………………………………………….…….. 51-55
VIII. APPENDICES ………………………………………………………..… 56-62
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LIST OF FIGURES
Figure 1 Growth of P.bahamense in nutrient replete medium
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Figure 2 Growth and toxin production of Pyrodinium bahamense at
various stages of growth
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Figure 3 Growth and toxin production of Pyrodinium bahamense at
varying salinity conditions
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Figure 4 Growth and toxin production of P. bahamense at varying
temperature conditions
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Figure 5 Growth and toxin production of P. bahamense at varying
salinity- temperature conditions
55
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APPENDICES
Appendix 1 Paralytic Shellfish Toxin Profiles of Pyrodinium
bahamense var. compressum cells
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Appendix 2 Chemical structure of PSP toxins
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Appendix 3 PPaarraallyyttiicc SShheellllffiisshh PPooiissoonniinngg ((PPSSPP)) ccaasseess iinn tthhee PPhhiilliippppiinneess
((11998833--22000000)) NNaattiioonnaall RReedd TTiiddee TTaasskk FFoorrccee--DDeeppaarrttmmeenntt ooff
HHeeaalltthh ((NNRRTTTTFF--DDOOHH))
Appendix 4 F/2 Medium
58
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Appendix 5 Thermal Gradient Device
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Appendix 6 Chromatograms obtained from a PSP standard mixture
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Appendix 7 HPLC system used for PSP determination with ion-pair
chromatography, chemical post-column oxidation and
fluorescence detection
62
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INTRODUCTION
The occurrence of harmful blooms in the Philippines caused by Pyrodinium
bahamense var. compressum has caused several Paralytic Shellfish Poisoning (PSP)
cases and loss in the aquaculture industry. The public health significance of this
organism can be correlated with the production of a potent group of neurotoxins
collectively known as saxitoxins, which varies among isolates. These toxins are
being accumulated by filter feeding bivalves which cause paralytic shellfish poisoning
in humans upon consumption of these contaminated shellfishes. Since its first
occurrence in the country in 1983, the organism continues to spread in Philippine
waters affecting new areas. However, few studies have been done on P. bahamense
var. compressum despite its public health and economic importance due to failure of
establishing laboratory cultures of the species (Blackburn and Oshima 1989). So far
the only reported cultures were from Palau, Malaysia and Philippines (Harada et al.
1982; Usup et al.1995; Corrales and Hall 1993). Establishment of cultures allow the
enumeration of toxins produced by a particular P. bahamense isolate since the toxin
composition may vary between species.
Despite the difficulty in culturing the organism, isolates of P. bahamense var.
compressum from the Philippines was successfully cultured in the laboratory
(Corrales and Hall 1993). Studies on the life history and aspects of the organism’s
biology have already been conducted but no studies have been done on the effects of
environmental conditions on the toxin content and composition of P. bahamense
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isolated in the Philippines. The only reported study of this kind on P. bahamense was
that of Usup et al. (1995) using a Malaysian isolate.
This pioneering work which is part of a project of Dr. Rhodora Azanza funded
by Commission on Higher Education (CHED) entitled “ Scaling-up of Pyrodinium
var. compressum cultures” aimed to determine the effects of varying salinity and
temperature conditions on the growth and toxin production of P. bahamense
isolated in the Philippines. Results on the growth and toxin production of a nutrient
replete laboratory culture of P. bahamense and the effects of varying salinity and
temperature conditions are presented in this study. This study is important in order to
understand the physiology of the organism and in predicting red tide bloom
occurrences. This could also be used as an important biochemical marker to
distinguish between geographical isolates as an identification tool in the study of
interrelationships between different and among P. bahamense var. compressum
isolates .
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REVIEW OF LITERATURE
Taxonomy
The thecate dinoflagelle Pyrodinium bahamense Plate var. compressum was
first described from the Bahamas in the Atlantic Ocean (Plate 1906). Samples from
the Persian Gulf examined by Böhm (1931) were shown to have a compressed
structure. Detailed morphology of the organism was studied by Taylor and Fukuyo
(1989). Steidinger et al. (1980) established two varieties of Pyrodinium bahamense,
var. compressum and var. bahamense from Tropical Pacific and Atlantic vegetative
cell samples, respectively. Both varieties were observed to have the same Kofoid plate
pattern of (P0Pi),4’, 6”, 6c, 8s, 6”’, 2”” (Balech 1985). Pyrodinium bahamense var.
compressum forms chains and appear to be anterio-posteriorly flattened. It is observed
to be confined in the tropical Pacific and produces PSP toxins. On the other hand,
variety bahamense is found to be non-chain forming, non-toxic and more localized in
the tropical Atlantic region.
Pyrodinium Blooms
Pyrodinium bahamense var. compressum has been reported as the main
causative organism causing Paralytic Shellfish Poisoning in (PSP) in Southeast Asia
affecting the Philippines (Estudillo and Gonzales 1984; Hermes et al. 1985; Corrales
et al. 1995), Brunei (Usup et al. 1989), Malaysia (Usup et al. 1993), Indonesia
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(Sidabutar et al. 1999) and other Indo Pacific countries (Maclean 1989) . Pyrodinium
bahamense started to bloom in the Philippines in June 1983 affecting Magueda Bay in
Samar which caused massive food poisoning cases due to consumption of PSP
contaminated shellfishes. The organism was observed to spread in Masinloc Bay,
Zambales in 1987 and Manila Bay in 1988. This incidents caused not only PSP
poisoning cases but also severe loss in the shellfish industry (Corrales and Maclean
1995). The organism continued to bloom from 1991-1998 which coincides with late
summer to early southwest monsoon. Blooms of P. bahamense is spreading in
Philippine waters infecting new areas like Palawan and Surigao in 1999. It has been
observed that previously affected areas like Samar and Leyte have been spared by
Pyrodinium blooms. To date, a total of 18 bays/areas have been affected by
Pyrodinium bahamense in the Philippines (Azanza and Taylor 2001).
Toxin Profiles
The presence of neurotoxins in this organism was first shown by Maclean
(1977) using standard mouse bioassay in isolates from Papua New Guinea. The
chemical nature of its toxins have been elucidated in a Palauan isolate using an ion-
exchange column and successive Thin Layer Chromatography (TLC) analysis (Harada
et al. 1983). The Palauan Pyrodinium isolate produces NeoSTX, STX, GTX5, GTX6
and a decarbamoyl saxitoxin (Harada et al. 1982). This has been the first recognition
of their occurrence in the natural environment (Harada et al. 1983). The carbamate
toxins which consists of STX, NeoSTX and GTX 1-4 are the most potent while the
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N-sulfocarbamoyl toxins (B and C) are the least potent. The dcSTX exhibits
intermediate specific toxicities and are generally less abundant in dinoflagellates
though there maybe important toxin components in certain bivalve species (Shumway
and Bricelj 1998; Sullivan et al. 1986). The significant pathway of each saxitoxin
derivative plays an important public health impact and varies among isolates under
varying growth conditions (Hall and Reichardt 1984).
Consequently, HPLC analysis of bivalves contaminated by Pyrodinium
isolated from Palau, Borneo and the Philippines have been found to contain the same
set of toxins present in the cultured cells from Palau (Oshima 1989). A different set of
toxins have been detected in Pyrodinium cells collected during a red tide outbreak on
the Pacific coast of Guatemala in 1987 which contained STX, NeoSTX, GTX2,
GTX3 and GTX4. No dcSTX or C-toxins were detected (Rosales-Loessener 1989).
However, toxin analysis of P. bahamense isolated from Sabah, Malaysia was found
to contain STX, NeoSTX, GTX5, GTX6 and dcSTX (Usup et al. 1997) different from
P. bahamense isolated from the Philippines which contained STX, dcSTX and
NeoSTX (Hummert et al.unpub.). Appendix 1 shows the toxin profiles of Pyrodinium
bahamense isolated from various regions (Azanza and Taylor 2001).
Few studies have been done on the effects of environmental conditions on the
growth and toxin production of P. bahamense. Most investigations on the effects of
salinity, light intensity, temperature and nutrient limitation in the growth and toxin
production were done on PSP-producing dinoflagellates Alexandrium spp. and
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Gymnodinium catenatum due to the difficulty in culturing the organism. The toxin
content in batch cultures of Alexandrium spp. was found to vary with growth stage
(Prakash 1967; Proctor et al. 1975; White and Maranda 1978; Oshima and Yasumoto
1979; Schmidt and Loeblich 1979). Increase in salinity caused an increase in the
toxin production (White et al. 1978), while a study conducted by Anderson and co-
workers (1990) showed no significant changes in the toxin content with varying
salinity conditions. The toxin content per cell increased as the temperature decreased
(Hall 1982; Ogata et al. 1987; Boyer et al. 1987; Anderson 1990). However, lower
light intensity caused an increase in toxin production (Ogata et al. 1987). Phosphorus
limitation promoted an increase in toxin content while a reverse effect was observed
with N- limitation (Anderson et al. 1990; Boyer 1987; Hall 1982; Maestrini et al.
2000). In Gymnodinium catenatum, no increase in toxin content was observed in
response to decrease in salinity (Flynn et al. 1996). The only study conducted on the
effects of various environmental conditions on the toxin production of P. bahamense
was done using a Malaysian isolate (Usup et al. 1994). Temperature showed a marked
effect on the toxin content which caused an increase in toxin content as temperature
was decreased. Similar effect was obtained in varying salinity conditions. However,
lowering the light intensity caused a decrease in the toxin content of the organism.
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Saxitoxin
Saxitoxins (STX) are produced by marine algae of the genus Alexandrium
spp., Gymnodinium spp., and Pyrodinium spp. (Simon et al. 1977; Van Egmond et al.
1992). Saxitoxin (C10H17N7O4, MW 299) is tricyclic molecule with the 1,2,3 and
7,8,9- guanidinium moieties of saxitoxin possessing pKa’s of 11.3 and 8.2,
respectively. The 1,2,3-guanidino carries a positive charge at physiological pH while
7,8,9-guanidino group is partially deprotonated. Its polar nature allows it to readily
dissolve in water and alcohols but not in organic solvents. Saxitoxin is stable in
solution at neutral and acidic pH even at high temperature. However, alkaline
exposure oxidizes and inactivates the toxin.
The saxitoxins are a family of water soluble neurotoxins (tricyclic
tetrahydropurine derivatives) and are among the most potent toxins known (Lehane
2000). Its mode of action involves a reversible and highly specific block of ion
transport by the sodium channel in excitable membranes such as the nerve cell and
fiber muscles (Narashi 1988). More than twenty structurally- related PSP derivatives
have been identified so far from toxigenic dinoflagellates and bivalves contaminated
with PSP toxins (Oshima 1989). These toxins vary widely in their biochemical
pathways or biological activities. Saxitoxins can be grouped into carbamoyl or
carbamate toxins which is composed of saxitoxin (STX), neosaxitoxin (NeoSTX) and
gonyautoxins 1,2,3 and 4 (GTX 1-4); sulfocarbamoyl or sulfamate toxins which
consists of gonyautoxins 5 and 6 (GTX5 and GTX 6) and its fractions (C1-C4); and
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decarbamoyl saxitoxins (dcSTX) (Lehane 2000). Appendix 2 shows the chemical
structure of PSP toxins.
Paralytic Shellfish Poisoning (PSP)
Paralytic Shellfish Poisoning is a food-borne illness caused by the
consumption of shellfish contaminated by PSP toxins. Shellfish concentrate these
toxins in their digestive tissues with considerable reduced amounts accumulated in
other tissues (Lee et al. 1987; Bauder et al. 1996). Several species of dinoflagellates
like Protogonyaulax spp., Gymnodinium catenatum and Pyrodinium bahamense are
the primary producers of these toxins (Taylor 1985). This type of poisoning causes
both gastro intestinal and neurologic symptoms with an onset time between 0.5 to 12h
after ingestion of contaminated shellfish. A slight perioral tingling progressing to
numbness which spreads to face and neck is usually observed in moderate cases.
However, in severe cases, these symptoms spread to extremities with incoordination
and respiratory difficulty. Medullary disturbances exhibited by difficulty in
swallowing, throat constriction, speech incoherence or complete loss of speech as
well as brain system dysfunction are also observed in severe cases. A complete
paralysis and death from respiratory failure within 2-12 hours may occur in very
severe cases in the absence of ventilatory support. After 12 hours, the victim starts to
recover gradually without any residual symptoms within a few days regardless of
severity (Bower et al. 1981; Halstead 1988). An oral dose of 1 to 4 mg (53 to 20 13
MU) in humans can cause death depending upon the age and physical condition.
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Other symptoms include headache, dizziness, nausea, vomiting, rapid pain and
anuria. There is no loss of conciousness and the reflexes are unaltered except maybe
pupillary size and sight may be temporarily lost.
Contaminated shellfish samples are usually analyzed by mouse bioassay
(AOAC 1980) but it can not distinguish between tetrodotoxin and other PSP toxins.
Radioimmunoassay and indirect ELISA have been developed for STX but not all PSP
toxins are identified (Carlson et al. 1984). The use of chemical method such as
HPLC have successfully detected all the saxitoxin derivatives (Sullivan et al. 1983
and Halstead 1988).
In the Philippines, the reported PSP cases and deaths were 2,111 and 117,
respectively, since Pyrodinium red tides occurred in 1983 (Appendix 3). In view of
the continuing threats of Pyrodinium red tides, water samples and shellfishes are
routinely analyzed to provide early warning signals on the presence of the organism
and to inform the public. A National Red Tide Task Force which is composed of
various government agencies has been established to monitor and manage red tide
occurrences.
PSP Toxin Analysis
Paralytic shellfish toxins are usually analyzed using biological and chemical
methods. Biological method usually include mouse bioassay (Hollingworth et al.
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1990; AOAC 1980) which is based on the pioneering work of Somer and Meyer
(1937). The enzyme linked immuno-sorbent assay (ELISA) (Chu and Fan 1985) is
also used to analyze the toxicity of shellfish though most shellfish monitoring
programs worldwide are based on mouse bioassay. However, analysis of PSP toxins
by mouse bioassay has several disadvantages such as negative interference from high
salt concentrations (Scahntz et al. 1958), insensitivity with a detection limit of only 1
MU/ml and 20% imprecision error and the need to maintain a mouse colony and
ethical objections against animal testings (Oshima 1989). A recent study by Park and
co-workers showed excessive variability from shellfish samples which have high
levels of toxicity. A receptor binding assay for detecting toxicity in shellfish and
algal extracts that is based on the high affinity reaction between PSP toxins and the
voltage-gated sodium channel was developed as an alternative to live animal testing
(Doucette et al.1997). The efficiency of the assay was later improved by incorporating
the microplate scintillation technology used in drug discovery studies (Doucette et al.
1999). The fact that the saxitoxins bind reversibly to its biological receptor, the
voltage-dependent sodium receptor channel, with binding affinity proportional to its
toxic potency makes the basis of the receptor binding assay for saxitoxin. This assay
is carried out by incubating the known receptor to the toxin in the presence of a
radiolabeled analog which together form a radiolabeled receptor-toxin complex. With
the addition of unlabelled toxin in the form of toxin standard or unknown sample in
the incubated mixture, the unlabeled toxin competes with the radiolabeled toxin for
the receptor, forming unlabeled complex. The amount of radiolabeled complex
formed in this mixture is quantified by liquid scintillation counting. With increasing
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amounts of unlabeled toxin, the amount of radiolabeled complex decreases relative to
the amount of radiolabeled complex formed in the absence of unlabeled toxin. The
amount of toxin present in an unknown sample is then quantified through a
competition curve between labeled and unlabelled toxin for the receptor (Van Dolah
1996). A radiometric receptor binding assay was set up recently in the Philippine
Nuclear Research Institute (PNRI) to complement the current live mouse bioassay
method as a component of the International Atomic Energy Agency (IAEA) HAB
project support. This method is based on the binding competition between the toxins
in shellfish samples or standard and 3H-labelled saxitoxin to the receptor sites.A
radiotracer, tritium labeled saxitoxin is used to measure the quantity of saxitoxin in
the sample bound to the receptor sites.
Alternatively, chemical procedures have been developed in which PSP toxin
fractions were separated. Chemical methods are usually based on ion-pair
chromatographic HPLC separation on different columns with post-column (Sullivan
1988; Oshima et al. 1989; Luckas 1987) or pre-column oxidation (Lawrence and
Menard 1991; Janecek et al. 1993) and fluorescent detection. The advent of reliable
HPLC methods for PSP toxin analysis has enabled detailed comparisons of toxin
composition among isolates (Oshima et al. 1989; Sullivan and Wekel 1987). Different
HPLC methods with different ion-pair reagents and RP-phases have been introduced
for analysis of PSP toxins since early 1980’s ( Sullivan and Wekel 1987; Lawrence
and Menard 1991; Oshima 1995; Hummert et al.1997). However, Oshima’s method
is more widely accepted because it renders determination of nearly all known PSP
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toxins. However, it needs three independent isocratic runs necessary to separate C
toxins, GTX toxins and NEO/dcSTX toxins in one chromatographic run. This method
was proven to be constant, rapid and advantageous for separation of dcSTX and
STX which is especially important for correct estimation of PSP toxicity of shellfish
samples (Hummert et al. 1997). However, this method is unable to separate GTX 1
and GTX 4 thus it can not give an exact toxin profile. Yu et al. (1998) modified
Hummert’s method to improve the separation of GTX 1 and GTX 4. The new
method is capable of determining all PSP toxins without interference (Appendix 4).
Separation of PSP toxins is significantly improved as compared to Hummert’s
method where GTX 1 and GTX 4 were now separated. These current HPLC
protocols use post-column chemical reaction system to oxidize the saxitoxin ring
system in order to form a fluorescent chromophore. However, this oxidation is
sensitive to changes in the flow rate, temperature, pH and the age of reagents. Boyer
et al. (1999) developed an HPLC-electrochemical oxidation system (HPLC-ECOS)
to eliminate the oxidation problem by utilizing an electrochemical cell. This system
comprises the HPLC pumping system and column, mobile phase preparation,
electrochemical oxidation cell and sample preparation. However, this technique has
problems associated with the care and maintenance of the oxidizing electrodes. These
chemical methods have an advantage of high sensitivity but also had disadvantages of
being labor intensive, time-consuming and expensive. Gerdts et al. (2002) developed
a simple Fast Fluorimetric Assay (FFA) for the detection of saxitoxin in algal
cultures and natural plankton samples based on the fluorimetric method developed
by Bates and Rapaport (1975) whereby the non-fluorescent saxitoxin molecule is
20
oxidized to fluorescent purine derivative by H2O2 treatment . Results of the assay was
correlated to HPLC results and showed to be significant for most of the carbamoyl
saxitoxins.
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MATERIALS AND METHODS
Growth Curve of Pyrodinium bahamense
Cell Counts
Pyrodinium bahamense var. compressum clone (PBC-MZ-061593) was
isolated from Bamban Bay, Zambales during an outbreak in June 1993. The cultures
were routinely maintained in the laboratory of RV Azanza at the Marine Science
Institute, University of the Philippines, Diliman in sterile seawater (30ppt) enriched
with F/2 medium (Guillard and Ryther 1962) at 24±2°C, 150 uEm-2s-1 under a 12:
12 h light : dark (L:D) cycle. Cells were subcultured every two weeks in fresh F/2
medium (Appendix 4).
The growth curve of the organism was determined using 500 ml nutrient
replete F/2 medium with an initial cell concentration of 100 cells/ml. Five ml
duplicate samples were withdrawn every 3 days for cell counting and preserved with
Lugol’s iodine. Samples withdrawn were replaced with sterile seawater with F/2
media. One ml from each sample was obtained for cell counts using Sedgewick
Rafter Chamber in a Zeiss light compound microscope under 200x magnification.
Cells were sampled until the cultures reached the death phase of growth.
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Growth Stages
Growth, toxin production and chlorophyll a content of Pyrodinium bahamense
at various stages of the life cycle were analyzed in 500 ml cultures at 24±2°C, 150
uEm-2s1 under a 12: 12 h light : dark (L:D) cycle. An initial cell concentration of 100
cells/ml were maintained in all the cultures used. Each 500 ml culture was harvested
after 4, 7,14, 21, 28, 35 and 43 days and were analyzed for the parameters indicated.
Cell Counts
The cell counts of each 500 ml culture harvested at various stages of growth
were analyzed. Two 5 ml duplicate samples were obtained upon harvest and were
fixed with Lugol’s iodine solution. One ml from each sample was obtained for cell
counts using Sedgewick Rafter Chamber in a Zeiss light compound microscope under
200x magnification.
Toxin Analysis
For toxin analysis, 100 ml cell samples were obtained from each 500 ml
culture harvested at various stages of the life cycle. Cells were filtered in 0.2 um
nitrocellulose membrane (Whatman), wrapped with aluminum foil and kept frozen
for HPLC analysis which was done at Friedrich Schiller University, Jena, Germany.
Chlorophyll a analysis
23
Four hundred ml each of P. bahamense cultures harvested at various stages of
growth were analyzed for chl a content as described by Strickland and Parson (1972).
Cells were filtered in 0.5 µm nitrocellulose membrane (Whatman) and 3 to 5 drops of
MgCO3 solution were added to preserve the cells. Filtered cell samples were placed in
glass test tubes wrapped with carbon paper and extracted in 10 ml 90% acetone. The
mixture was thoroughly mixed to ensure that the filter paper is completely dissolved
in the solution and refrigerated overnight. Samples were then centrifuged at 1500
rpm for 8 minutes (Symantic) and analyzed using a spectrophotometer (Genesis 2) at
various wavelengths (750, 665, 664, 647 and 680 nm). The amounts of cholorophyll a
in the sample were calculated using the given equation below (Strickland and Parson
1972).
(Ca) Chlorophyll a = 11.85E664-1.54E647-0.08E630
where: E stands for the absorbance at different wavelengths obtained from the
corrected 750nm reading.
Salinity effects
The effects of salinity on the growth and toxin production of P. bahamense
were studied using natural seawater (30 0/00 ) enriched with F/2 as the basal medium.
A range of salinities (24, 26, 28, 30, 32, 36 0/00 ) in the medium was established by
dilution with deionized water and sodium chloride (NaCl) to get the desired
salinities. P. bahamense cells were grown and acclimatized over three transfers to
24
each salinity range and maintained at 28±2°C, 150µEm-2s-1 under a 12: 12 h L:D
cycle. Exponentially growing cells in each salinity condition were subcultured in
duplicate 100 ml culture flasks. An initial cell concentration of 100 cells/ml were
maintained for all the set-ups. Cells were harvested during the early exponential
growth phase. Duplicate samples of 5 ml each were withdrawn for cell counts and
fixed with Lugol’s iodine. The rest of the culture was filtered for toxin analysis using
HPLC.
Temperature effects
Temperature effects on the growth and toxin production were studied using a
thermal -gradient device (Watras 1982). The device is made up of 127cm x 20cm x
63cm wooden plywood frame with an aluminum sheet warmed by two incandescent
bulbs in one end (Appendix 5). A 40-W fluorescent fixture was fitted with chains on
top of the wooden frame. Desired light intensity was obtained by adjusting the height
of the fluorescent lamp. The aluminum plate was attached at the ends and rested on
the sides of the frame. The side of the frame was provided with an access door for the
bulbs which were mounted on a vertical adjustable bracket and a vent opening on the
“cool” end sufficiently large to ensure that the gradient plate remains close to room
temperature. The temperature of the gradient bar was controlled by changing the
wattage of the bulbs. The apparatus accommodated three rows of 125 ml flask at each
desired temperature in the gradient bar. Temperatures were measured using an
indoor/outdoor thermometer with a probe .
25
A temperature range of 26 to 38 C was established by adjusting the heat from
the incandescent bulb on one side. Cultures at each temperature range were allowed
to acclimatize over three transfers and maintained at 150 µEm-2s-1. Exponentially
growing cells were transferred in 100 ml culture flasks. Duplicate samples were
maintained at each temperature range. Cultures were grown under continuous
illumination and were harvested in the early exponential growth phase. Samples were
withdrawn for cell counts and toxin analysis as described above.
Combined salinity-temperature effects
The combined effects of varying salinity and temperature were studied in 100
ml cultures using the thermal gradient bar. Salinity and temperature combinations
used were 26 0/00 -26 C, 26 0/00 -32 C, 26 0/00 - 35 C, 30 0/00 - 26 C, 30 0/00 - 32 C,
30 0/00 - 35 C, 36 0/00 -26 C, 36 0/00 -32 C and 36 0/00 - 36 C. Cultures were
acclimatized at each salinity-temperature condition over three transfers.
Exponentially growing cells were subcultured in duplicate 100 ml flasks. An initial
cell concentration of 100 cells/ml were maintained for all the set-ups. Cultures were
maintained under continuous illumination and were harvested during the early
exponential stage of growth. Duplicate subsamples of 5 ml each were withdrawn for
cell counts and the rest of the cultures were filtered for toxin analysis.
HPLC Analysis of PSP toxins
26
Chemicals
Standard solutions of STX, NeoSTX, GTX 1, GTX2, GTX3, and GTX4 were
purchased from the National Research Council, Canada, Marine Analytical Standard
Program (NRC-PSP-1B) Halifax NS, Canada. The standard solution GTX2 and
GTX3 contained dcGTX2 and dcGTX3 as minor components but the exact contents
of these toxins were not given. DcSTX was provided by the European Commission
(The Community Bureau of Reference, Brussels). All solvents used were HPLC
grade, where acetonitrile was from Riedel-de Haen, Geel, Belgium, tetrahydrofuran
was from ROTH, Carl ROTH GmbH, Germany. Octane sulfonic acid was purchased
from Sigma, Germany. All other chemicals used were analytical grade. Water used for
HPLC was purified with Millipore-QRG ultra pure water system (Millipore, Milford,
USA).
Apparatus
The chromatographic system consisted of an AS-400 intelligent autosampler
and L-6200A intelligent pump (both Merck-Hitachi, Darmstadt, Germany), two LC-
9A pumps (Shimadzu, Duisburg, Germany) used for delivery of post column reaction
solutions, an FR551 Fluorescence detector (Shimadzu), an 1 ml CRX390 post
column reaction unit (Pickering Laboratories, Mountain View, CA, USA) and a D-
6000 HPLC- manager (Merck-Hitachi).
27
HPLC Analyses
The analysis of PSP toxins was done based on the optimized Liquid
Chromatography (LC) method developed by Yu et al. 1998. This method was
established from the method for the separation of PSP toxins developed by Hummert
(Hummert et al. 1997). In this new method all the PSP toxins were determined
without any interference. Appendix 6 shows the chromatograms obtained with the
original Hummert’s method and the optimized method.
The separation of PSP toxins with the modified method was significantly
improved as compared to the original method, i.e., The GTX1 and GTX4 toxins were
baseline separated whereas with the original method, these PSP components were co-
eluted. HPLC analysis was done at Department of Chemistry, Friedrich-Schiller
University, Jena, Germany. Appendix 7 shows the HPLC system used for PSP
determination with ion pair chromatography, chemical post column oxidation and
fluorescent detection.
Extraction of PSP toxins
28
Filtered P. bahamense samples were extracted using 50:50 (v/v)
methanol:water. One ml of solvent was used to extract the toxins from the filtered
sample. Extracted sample was mixed in a vortex for 1 min and homogenized in
ultrasonic bath for 10 min. Sample was then soaked in the mixture for 30 min and
homogenized again in ultrasonic bath for 10 min. Homogenized sample was
centrifuged at 3000 rpm for 10 min and the supernatant was filtered with single-use
syringe filters (0.45µm, polypropylene).
Hydrolysis of PSP toxins containing extracts
The extracts containing PSP toxin were mixed with 150 ul of acetic acid
(0.03N) and 37 l 1M HCl, vortexed for 20 sec and hydrolyzed by heating for 15 min
at 90°C. Each sample was cooled down to room temperature and mixed for 30 sec.
The mixture was neutralized with 76 l 1N sodium acetate solution and mixed for 30
sec.
HPLC-FD determination of PSP toxins using ion-pair chromatography and post
column oxidation
About 10-20 l of hydrolyzed sample was injected in the HPLC column
(Luna 5 µm RP-C18 (250mmx4.6 mm) Phenomenex). Three different mobile phases
were used : 1) 98.5% 11 mM octanesulfonic acid (sodium salt) and 40 mM
phosphoric acid adjusted to pH6.9 with NH3 and 1.5% tetrahydrofurane, 2) 83.5 % 11
29
mM octanesulfonic acid and 40 mM phosphoric acid, adjusted to pH 6.9 with NH3
and 15% acetonitrile and 1.5% tetrahydrofurane, 3) 98.5% 40mM phosphoric acid
adjusted to pH 6.9 with NH3 and 1.5% tetrahydrofurane. A flow rate of 1ml /min was
used with a column temperature of 25°C (3°C). Post column derivatization of the
toxins was done by oxidation in 10.0 mM periodic acid and 550 mM NH3 solution
(0.3ml/min), acidified in 1 mM nitric acid (0.4 ml/min) at 50°C. The fluoromonitor
was set at 330 nm and 395 nm for elution and emission wavelengths, respectively.
Toxin Quantification
Quantification of the carbamoyl toxins was carried out by comparing the
peak areas obtained for sample extracts with those peaks obtained after injection of
standard solutions. Hydrolyzed and non-hydrolyzed sample extracts were injected for
quantification of all N-sulfocarbmoyl toxins (inclusive of GTX5/B1 and GTX6/B2)
by calculating the peak height increases for related carbamoyl toxins during
hydrochloric acid treatment (B1 to STX, B2 to NEO, C1 to GTX2, C2 to GTX3, C3
to GTX1 and C4).
RESULTS
30
The growth curve of P. bahamense in nutrient-replete batch cultures is
shown in Figure 1a. Early exponential stage of growth was achieved after 7 days in
culture. Stationary phase of growth was reached after 28 days. Growth started to
decline after 35 days and deaths were observed after 43 days. Chlorophyll a content
of the culture showed an increasing trend as the culture reached the exponential stage
of growth ranging from 1.80-7.38 µg/L (Fig 1b) which coincides with an increase in
cell number of P. bahamense cells in culture. Chlrophyll a content reached a constant
value as the cultures approached the stationary phase of growth. Under normal
growth in F/2 medium, specific growth rate of 0.2 div d-1 was observed during the
early stages of growth and increased drastically from 0.2 to 0.3 div d-1 as it reached
the death phase (Fig 2a). The toxin content reached a peak of 298 fmol cell-1 during
the mid-exponential stage (Fig 2b). A rapid decline in the toxin content was observed
as the culture reached the stationary phase and remained constant with a toxin content
of 54 fmol cell-1. The toxin composition of this isolate was shown to produce only
three sets of toxins composed of STX, dcSTX and B1 (Fig 2c). STX made up most of
the toxin produced by this isolate which was composed of about 90% of the total
toxin. NeoSTX, GTX 1-6 and C toxins were not produced during the entire growth
cycle of the organism. Though a marked variation in toxin content was observed, the
toxin composition of P. bahamense remained constant until stationary phase.
However, a 20 % decline in STX mole % toxin cell-1 value was observed and 15%
increase dcSTX mole % toxin cell-1 value were observed as the culture reached the
death phase.
31
This P. bahamense isolate was able to grow at salinities ranging from 26 to
36 0/00 with an optimum growth at 30 0/00 or higher. Specific growth rate increased
with an increase in salinity of the culture medium ranging from 0.2 to 0.4 div d-1. The
growth rate was highest at 36 0/00 which reached up to 0.4 div d-1 (Fig 3a). The toxin
content was observed to be highest at 30 0/00 (260 fmol cell-1) and decreased to about
60 fmol cell-1 at higher salinity (36 0/00 ) (Fig 3b). The decline in toxin content with
increased salinity coincided with an increase in specific growth rate of the organism.
Salinity has a slight effect on the toxin composition. A decreasing trend in mole %
toxin cell-1 of STX (from 80 mole % toxin cell-1 to 65 mole % toxin cell-1) was
observed with increased salinity while dcSTX increased from 15 mole % toxin cell-1
to 32 mole % toxin cell-1 (Figure 3c). B1 toxins remained constant in all the salinity
variations comprising about 6 mole % toxin cell-1 of the total toxin content.
Under various temperature culture conditions, P. bahamense was able to grow
from 23 to 36C with an optimum growth at 25C. Specific growth rate was lower
with increased toxin content. Lowest growth rate was observed at 25°C which is
about 0.22 div d-1 (Fig 4a). Highest growth rate was achieved at 0.4 div-1 at 28C. A
marked increase in toxin content (376 fmol cell-1) was observed with 7C decrease in
temperature (Fig 4b). Toxin content were observed to be constantly lower (80 to 116
fmol cell-1) at higher temperature (32 to 36C). Though P. bahamense was able to
grow at 36 C. The toxin composition profile showed that STX made up to 85 to 98
mole % toxin and the remaining were B1 and dcSTX (Fig 4c). No dcSTX was
produced from 25 to 34 C. However at 36 C, STX dropped by 50 mole % toxin
32
cell-1 while large amounts of dcSTX and B1 were produced amounting to 41 and 17
mole % toxin cell-1 , respectively.
Combined effects of salinity and temperature showed that P. bahamense was
not able to grow at low salinity and low temperature (260/00 -28C). Optimum growth
was observed at higher salinities in all temperature conditions. Specific growth rate
ranged from 0.2 to 0.4 div d-1 which peaked at 30 0/00 in all the temperature
conditions (Fig 5a). The toxin content obtained was in the range of 101 to 287 fmol
cell-1 in various salinity-temperature culture combination. However, an increase in
toxin content showed a decline in the growth rate. The toxin content was highest at
300/00 and 26C amounting to 665 fmol cell-1 (Fig 5b). Combined effects of salinity
and temperature showed similar results with varying temperature. STX composed
about 80 to 99 mole % toxin cell-1 (Fig 5C). Absence of dcSTX was observed in most
of the salinity–temperature culture condition except at 260/00 -32C and 300/00 -28C
which produced 14 and 7 mole % toxin cell-1, respectively.
33
DISCUSSION
Culture of P. bahamense
The difficulty in culturing P. bahamense has limited attempts to study the
physiology of this important species. Many of the studies on the toxin content and
composition of PSP producing dinoflagellates have been done on Alexandrium and
Gymnodinium species (Anderson et al. 1990; Boyer et al. 1987; Cembella et al. 1987;
Hall 1982; Flynn 1996). The Philippine P. bahamense isolate was successfully
cultured initially using F/20 medium in 1991. Different culture media were used to
culture this isolate. Cultures in sterile seawater, F/2 and F/4 media did not last for
more than a week while cultures in F/10 and F/20 media lasted for two and three
months, respectively (Corrales and Hall 1993). In this study, the growth of Philippine
P. bahamense isolate in nutrient-replete batch culture using F/2 medium was observed
to last for six weeks. Cells started to grow vigorously after 3 days and rapidly
declined after 35 days. However, batch cultures of Malaysian P. bahamense isolate
grown in ES-1 medium was observed to reach prolonged stationary phase of growth
within the 30-day culture period (Usup et al. 1995).
Laboratory cultures of P. bahamense (PBC-MZ061593) subjected to varying
salinity conditions were able to grow at salinities ranging from 26 to 36 0/00.
Optimum growth was achieved at 36 0/00. Similar results were obtained in Malaysian
P. bahamense isolate which grew at 20 to 35 0/00. Field data during P. bahamense
blooms suggested that this species prefer to grow at high water salinity. In the
34
Philippines, blooms occurred in waters of 31 0/00 or higher salinities (Corrales and
Hall 1983), which coincide with the data obtained from the study conducted in the
laboratory. In Malaysia, blooms of the organism were observed in waters of salinities
30 0/00 or higher (Usup et al. 1989 ) while in Papua New Guinea blooms occurred in
waters of 28 0/00 or higher salinities (Maclean 1976).
The organism was able to grow at 24 to 36 C in the laboratory with optimum
growth at 26 C. A decrease in cell growth was observed with increase in
temperature. P. bahamense isolate from Malaysia showed that the temperature limits
for growth are 22 to 34 C with an optimum growth at 28 C. Field data showed that
seawater temperature in natural habitat of P. bahamense ranges from 25 to 31 C
(Usup et al.1995).
Toxin Composition
In this study, the Philippine P. bahamense was found to contain only three sets
of toxins composed of STX, dcSTX and B1. STX made up 90 mole % toxin cell-1 of
the total toxin produced and the remaining 10 mole % toxin cell-1 was divided
between dcSTX and B1. Isolates from Palau in the south Pacific are abundant in
GTX4 and GTX 5, STX and Neo with low percentage of dcSTX (Oshima et al. 1984;
1987). The Malaysian isolate however was found to produce 5 sets of toxins
composed of Neo, GTX 5, STX, GTX 6 and dcSTX. Neo and GTX 5 are the major
toxins produced comprising about 80 mole % toxin cell-1 under all the condition
35
studied (Usup et al. 1995). There are some indications of biogeographical variation in
toxin profile of P. bahamense. Toxins detected in cells during a bloom of the
organism in Guatemala contain STX, Neo, GTX 2, GTX 3, and GTX 4 without any
dcSTX (Rosales- Loessener et al. 1989). Most Pyrodinium analyzed so far are not
capable of producing C toxins.
In contrast to P. bahamense., significant regional variation has been observed
among Alexandrium populations providing genetic stability of toxin expression.
Alexandrium isolates from Alaska, British Columbia and Washington State contain
relatively high amounts of C1/ C2 and B1/B2 (Hall 1982, Cembella and Taylor
1985), while isolates from Atlantic Canada have conservative toxin composition
exhibiting less intra-specific and geographical variation (Cembella and Destombe
1996).
Isolates of G. catenatum exhibit an unusual toxin profile consisting mainly of
N-sulfocarbamoyl toxins (C1-C4, B1/B2) with no carbamate toxins produced
(Oshima et al. 1983; 1993). Isolates from Tasmania, Japan and Spain are
discriminated from each other by the presence of a novel component 13-
deoxycarbamoyl toxins, absence of C3/C4 toxins and high relative amounts of B1 and
B2, respectively. Singapore strains revealed a unique profile that was dominated by
the highly potent carbamate toxins, primarily GTX 1 and 4 with lesser amounts of
GTX 2, GTX 3, neoSTX and STX. No N-sulfocarbamoyl, decarbamoyl or deoxy-
decarbamoyl toxins dominate the toxin profiles of all other populations examined so
36
far. However, direct comparisons are difficult to make since Pyrodinium produces
fewer toxins than species of Alexandrium and Gymnodinium. These compositional
changes have important health implications due to significant differences in potency
between saxitoxins (Genenah and Shimizu 1981, Hall and Reichardt 1984) aside from
being a useful tool in studying interrelationship between dinoflagellate species and
populations (Cembella et al. 1987). However, toxin compositional changes in a
single isolate occur under different conditions (Anderson 1990). In this study, the
toxin composition of nutrient- replete Philippine P. bahamense isolate in batch
cultures was found to be constant over time until prolonged stationary phase of
growth. A similar result was observed in P. bahamense Malaysian isolate. Boczar et
al. (1988) demonstrated toxin composition variability in A. tamarense and A.
catenella which showed compositional changes in old batch cultures that have been
in the plateau phase for several weeks. This represent an unusual physiological
condition reflecting differential catabolism of the various toxins with little relevance
to actively growing cells (Anderson et al. 1990). However, toxin compositional
changes were evident when P. bahamense was subjected to various environmental
conditions. A 20 mole % toxin cell-1 increase and decrease in dcSTX and STX,
respectively was observed in P. bahamense cultured at higher salinity (36 0/00). In this
case, the most accurate geographical comparison among dispersed isolates would be
based on the presence or absence of each toxin and not the relative concentration of
each toxin (Anderson et al. 1990). The cellular toxicity and toxin profiles have been
used as chemotaxonomic indicators among populations and species. The toxin
composition is a relatively conservative characteristic within an isolate or natural
37
population and can be used to evaluate genetic differentiation among species of
diverse geographical regions (Cembella and Taylor 1985, Cembella et al. 1987,
Anderson et al. 1984).
Cellular Toxin Dynamics
Under the normal growth culture condition in nutrient-replete batch culture,
the toxin content of P. bahamense was found to be low at lag phase and showed
peaks during the mid exponential phase. Toxin content started to decline as the
culture approached the early stationary phase of growth. Similar results were obtained
in nutrient-replete batch cultures of the Malaysian P. bahamense isolate where the
toxin content at mid-exponential phase was twice greater than late-exponential and
stationary phase of growth. These observations can be explained by the fact that
nutrients are not usually balanced during early exponential growth phase in batch
cultures due to nitrogen “upshock” when cells are newly transferred to a fresh
nutrient-replete medium (Flynn and Flynn 1996). During the late exponential growth,
CO2 depletion or nitrogen limitation occurs whereby the toxin is lost to daughter cells
faster than it is produced (Anderson 1990). Decrease in toxin content at the stationary
phase may be attributed to several factors such as leakage, cell lysis, decrease in the
rate of production, increased turnover or partitioning of toxin into daughter cells via
cell division (Cembella 1998). Physiological measurement showed useful relationship
between growth rate and toxin production in batch cultures due to production of
arginine, an amino acid precursor to saxitoxin production. Arginine was observed to
38
be very low when toxin content peaked at the exponential phase and increased
rapidly as toxicity declined approaching the death phase. The elevation of toxin
content during the early exponential phase of batch culture could be a result of the
conversion of excess free arginine into PSP toxins following the initial surge in
uptake of nitrogen (Usup et al. 1995).
An inverse relationship was observed between toxin content and division rate.
Growth rate was higher with a decrease in toxin content. This relationship has also
been demonstrated in several Alexandrium species subjected to various
environmental stresses in batch cultures (Anderson et al. 1990; Ogata et al. 1990;
Proctor et al. 1975) but not to all culture conditions (Anderson et al. 1990). In this
case, toxin accumulation is faster during the early stage of growth than toxin transfer
to daughter cells during cell division. The optimum condition in batch culture is
temporarily achieved and may be subjected to nutrient limitation or carbon dioxide
depletion (Anderson 1990). Contrary to these results, study on different isolates of A.
tamarense and sub strains at different growth rates showed no correlation between
growth rate and toxicity (Kodama 1990). Hence, an understanding of the relationship
between toxicity and growth rate is important in order to determine whether the
toxicity is directly dependent on environmental variations or indirectly affected by
environmental factors on growth rate (Parkhill et al. 1999).
Salinity Effects
39
The survival of P. bahamense in varying salinity conditions in this study was
found to range from 26 to 36 0/00 where highest toxin content was obtained at 30 0/00.
A drop in toxin content from 250 to 50 fmol cell-1 was observed as the salinity was
increased to 36 0/00 with an increase in growth rate to about 0.4 div day-1. In P.
bahamense, the enhanced Qt at the lowest salinity coincided with the lowest growth
rate (Usup et al. 1994). Combined effects of salinity and toxin production showed a
decrease in growth rate with increase in toxin production. Optimum toxin content was
obtained at 30 0/00 - 26°C.
Most of the toxigenic dinoflagellates are euryhaline species and it is unlikely
that salinity fluctuations either affect growth rate or toxin content per cell in nature
(Cembella 1998). Discrepancies on the results obtained in various studies regarding
the mean maximum rate for salinity-dependent growth are probably due to differences
in species or clones and not due to salinity fluctuations (White 1978). A study on A.
tamarense showed no significant effect on the growth rate within the salinity range of
20 to 30 0/00 (Cembella and Therriault 1989). The mean growth rate obtained was
similar to the results obtained in previous studies on estuarine clones in which the
optimum salinity was between 20 and 30 0/00 (Prakash 1967; White 1978 ; Watras et
al. 1982). However, toxin content was observed to increase with increasing salinity
in studies conducted in Gonyaulax excavata (Alexandrium fundyense) (White 1978).
Anderson et al. (1990) found no effects of salinity on the toxin content of A.
fundyense in acclimated cultures or those subjected to short-term changes in salinity.
40
In this study, toxin content was observed to decrease with increase in salinity. For the
Malaysian P. bahamense isolate, no elevation of toxin content was observed with
increasing salinity but there was a significant increase at low salinity.
Temperature Effects
The effects of varying temperature conditions showed that toxin content was
higher at lower temperature with lower growth rate. Results showed that the toxin
content of P. bahamense at 25°C peaks at 400 fmol cell-1 as compared to higher
temperature conditions though growth rate of the organism was high . In the case of
the Malaysian P. bahamense isolate, maximum change in toxin content increased
three fold from 200 to 600 fmol cell-1. Studies on Alexandrium species showed an
increase in toxin content as the growth temperature decreased ( Anderson et al. 1990;
Hall 1982; Ogata 1987). This may be the reason why apparent high cell toxicity of
Alexandrium populations are found at high latitudes (White 1986; Cembella et al.
1988). However, the Malaysian P. bahamense isolate has a Q10 of 1.46 for growth
rate for the temperature range of 22-32°C which is less than the average value of 1.8
obtained for several other phytoplankton species in batch culture (Eppley 1972;
Raven and Geider 1988). Metabolic reactions associated with enzymatic activity are
susceptible to the Q10 effect. The elevation of toxin content at low temperatures is not
only due to low division rates but also associated with other factors like turnover rates
of cellular component. Change in toxin profile was observed with increased
temperature. Gradual inversion of high ratio of B1/NEO toxin with increasing
41
temperature gradient was observed in the Malaysian P. bahamense isolate which
indicates a temperature-dependent process (Usup et al. 1994). A decline in STX
component while an increase in B1 and STX were observed in the Philippine
P.bahamense isolate during high temperatures. The elevation of toxin content at low
temperatures may not be simply due to low division rates but to other factors such as
turn over rates of cellular components (Usup et al. 1995). High arginine levels during
exponential growth was observed at low temperature cultures with elevated toxin
content. The low temperature may have inhibited protein synthesis thereby affecting
the growth rate since the protein level of these cells are low, thereby resulting in a
surplus of arginine within the cell that could be used for toxin synthesis (Anderson
1990).
42
CONCLUSION
The toxin profile of Philippine P. bahamense isolate was shown to be
composed of three sets of toxins, namely STX, dcSTX and B1 which was found to be
different from P. bahamense isolates from Palau, Malaysia and Guatemala. This
difference can be used as a biochemical marker to differentiate species of
P.bahamense in different geographical regions and study their interrelationships.
Highest toxin content was obtained during the mid-exponential phase of growth.
However, toxin content was observed to decrease with increase in salinity. Highest
toxin content was found in cultures maintained at 30 0/00. Lower temperature (26°C)
promoted an increase in toxin content of the organism. These observations are
important contributions in the understanding of the bloom dynamics of this
important PSP producing organism.
43
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58
0
200
400
600
800
1000
0 4 7 14 21 28 35 43 50
Ce
ll c
ou
nts
(ce
ll/m
l)
0
1
2
3
4
5
6
7
8
0 4 7 14 21 28 35 43 50
ug/L
Days
Figure 1: a) Growth of P.bahamense in nutrient replete medium b) Chlorophyll a
content of P. bahamense at various stages of growth cycle
a
b
59
0
0.1
0.2
0.3
0.4
0.5
0 4 7 14 21 28 35 43 50
k (
div
-d)
0
50
100
150
200
250
300
350
400
0 4 7 14 21 28 35 43 50
Toxin
cell
-1(f
mol)
0
20
40
60
80
100
120
140
0 4 7 14 21 28 35 43 50
Mo
le %
to
xin
ce
ll-1
%STX
%dcSTX
%B1
Days
Figure 2 : Growth and toxin production of Pyrodinium bahamense at
various stages of growth a) specific growth rate b) Cellular toxin content c)
Cellular toxin composition
a
b
c
60
0
0.1
0.2
0.3
0.4
0.5
0.6
24 26 30 32 36 38
k (
div
d-1
)
0
50
100
150
200
250
300
24 26 30 32 36 38
To
xin
cell-1
(fm
ol)
0
10
20
30
40
50
60
70
80
90
24 26 30 32 36 38
Mo
le %
to
xin
cell
-1
%STX
%dcSTX
%B1
Figure 3: Growth and toxin production of Pyrodinium bahamense at varying salinity
conditions a) Specific growth rate b) Cellular toxin content c) Cellular toxin
composition
Salinity
a
b
c
61
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
20 23 25 28 30 32 34 36 38
k (
div
d-1
)
0
100
200
300
400
500
20 23 25 28 30 32 34 36 38
Toxin
cell-1
(fm
ol)
0
20
40
60
80
100
120
20 23 25 28 30 32 34 36 38
Mole
% t
oxin
cell-1
%STX
%dcSTX
%B1
Figure 4: Growth and toxin production of P. bahamense at varying temperature
conditions a) Specific growth rate b) Cellular toxin content c) Cellular toxin
composition
Temperature
a
b
c
62
0.00
0.10
0.20
0.30
0.40
0.50
26ppt-
26°C
26ppt-
32°C
26ppt-
35°C
30ppt-
26°C
30ppt-
32°C
30ppt-
35°C
36ppt-
26°C
36ppt-
32°C
36ppt-
35°C
k ( d
iv d
-1)
0
100
200
300
400
500
600
700
800
26ppt-
26°C
26ppt-
32°C
26ppt-
35°C
30ppt-
26°C
30ppt-
32°C
30ppt-
35°C
36ppt-
26°C
36ppt-
32°C
36ppt-
35°C
To
xin
ce
ll-1 (
fmo
l)
B
0
20
40
60
80
100
120
140
26ppt-
26°C
26ppt-
32°C
26ppt-
35°C
30ppt-
26°C
30ppt-
32°C
30ppt-
35°C
36ppt-
26°C
36ppt-
32°C
36ppt-
35°C
Mo
le %
to
xin
ce
ll-1
%STX
%dcSTX
%B1
C
Salinity-temperature
Figure 5: Growth and toxin production of P. bahamense at varying salinity-temperature
conditions a) Specific growth rate b) Cellular toxin content c) Cellular toxin composition
a
b
c
63
Appendix 1 : Paralytic Shellfish Toxin Profiles of Pyrodinium bahamense var. compressum cells (Azanza et al. 2000)
Species
Origin
Toxin Compositions
Carbamate N-sulfocarbamoyl Decarbamoyl Reference
Pyrodinium Palau STX, NeoSTX GTX5, GTX6 dcSTX Harada et al. 1982
and 1983
Guatemala STX,NeoSTX GTX 2,GTX 3 and
GTX 4
Rosales-Loessener
et al. 1989
Malaysia STX,NeoSTX GTX 5, GTX 6 dcSTX Usup et al. 1994
Philippines a STX, NeoSTX - dcSTX Hummet et al.
unpub.
Perna viridis Philippines STX, NeoSTX GTX 5 dcSTX Oshima et al. 1989
a Pyrodinium bahamense isolated from the Masinloc,Zambales, Philippines
64
N
N
NH
NH
NH2
+NH
2
+
OH
OH
R2 R3
R1
R4
O
NH2 O
R4:
13
679
10
11
12
21
abbreviation:
STX:
NEO:
GTX:
dc:
do:
charge
Saxitoxin
Neo-Sax toxin
Gonyautoxin
Decarbamoyl
Deoxidecarbamoyl
(at pH 7.0)
= ++
= +
= 0
Carbamoyl N-
Sulfocarbamoyl
Decarbamoyl Deoxidecar
toxins toxins toxins bamoyl toxins
R1 R2 R3 R4: OCO-NH2 R4: OCONH-SO3- R4: OH R4: H
H H H STX B1 dcSTX doSTX
OH H H NEO B2 dcNEO -
OH OSO3- H GTX1 C3 dcGTX1 -
H OSO3- H GTX2 C1 dcGTX2 doGTX2
H H OSO3- GTX3 C2 dcGTX3 doGTX3
OH H OSO3- GTX4 C4 dcGTX4 -
Appendix 2: Chemical structure of paralytic shellfish poisoning toxins (Oshima 1989)
65
AAppppeennddiixx 33:: PPaarraallyyttiicc SShheellllffiisshh PPooiissoonniinngg ((PPSSPP)) ccaasseess iinn tthhee PPhhiilliippppiinneess ((11998833--22000000))
NNaattiioonnaall RReedd TTiiddee TTaasskk FFoorrccee--DDeeppaarrttmmeenntt ooff HHeeaalltthh ((NNRRTTTTFF--DDOOHH))
0
50
100
150
200
250
300
350
2000 1999 1998 1997 1996 1995 1994 1993 '92-
93
1992 1991 1990 1989 1988 1987 1983
Cases Deaths
66
Appendix 4: F2 Medium (Guillard, R.R.L. and Ryther, J.H. 1962)
1. NaNO3 150 g l-1 H20
2. NaH2PO4 10 g l-1 H2O
3. Trace Metals
CuSO4.5H2O 19.6 mg l-1 H2O
ZnSO4.H2O 44.0 mg l-1 H2O
CoCl2.6H2O 22.0 mg l-1 H2O
MnCl2.4H2O 360.0 mg l-1 H2O
NaMoO4.2H2O 12.6 mg l-1 H2O
4. Fe citrate
Ferric citrate 9.0 g l-1 H2O
Citric acid 9.0 g l-1 H2O
( Sterilize at 121°C for 15 min.)
5. Vitamins
Thiamine HCl 20.0 mg 100 ml-1 H2O
Biotin 0.1 100 ml-1 H2O
B12 0.1 100 ml-1 H2O
(Prepare fresh solution every 3 months)
NOTE: Refrigerate all stock solutions
Add 1 ml of each stock solution, except phosphate to 1 liter distilled water.
Phosphate must be sterilized separately from seawater to prevent precipitation.
Add 1 ml of phosphate stock solution to 3ml distilled water. Autoclave at
1210C for 15 min. After cooling, add phosphate aseptically to seawater
medium.
Alternatively dilute phosphate stock with 1 ml distilled water per flask.
Sterilize diluted phosphate solution and add aseptically.
To Prepare Medium f2
Prepare as medium f, but add 0.5 ml of each stock solution instead of 1.0 ml
of each.
Stock Solutions
To prepare Medium f
67
a)
b)
Appendix 6: Chromatograms obtained from a PSP standard mixture Hummert’s Method
b) Modified Method (Yu et al. 1998)