Download Paralytic Shellfish Poisoning Toxins: Biochemistry and Origin Masaaki Kodama

Aqua-BioScience Monographs Vol. 3, No. 1, pp. 1–38 (2010)
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Paralytic Shellfish Poisoning Toxins:
Biochemistry and Origin
Masaaki Kodama
Laboratory of Marine Biochemistry, Department of Aquatic Biosciences,
Graduate School of Agricultural and Life Sciences, The University of Tokyo
1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
e-mail: [email protected]
Abstract
Plankton feeders such as bivalves often become toxic. Human consumption of the toxic bivalve
causes severe food poisoning, including paralytic shellfish poisoning (PSP) which is the most
dangerous because of the acuteness of the symptoms, high fatality and wide distribution throughout
the world. Accumulation of PSP toxins in shellfish has posed serious problems to public health
and fisheries industry. The causative organisms of PSP toxins are known to be species of
dinoflagellates including those belonging to the genus Alexandrium, Gymnodinium catenatum
and Pyrodinium bahamense var. compressum. Bivalves accumulate PSP toxins during a bloom
of these dinoflagellates. Thus, the dinoflagellate toxins have been considered as being concentrated in bivalves through food web transfer. However, field studies on the toxin level of bivalves
in association with the abundance of toxic dinoflagellates could not support the idea. A kinetics
study on toxins by feeding experiments of cultured dinoflagellate cells to bivalves also showed
similar results, indicating that toxin accumulation of bivalves is not caused by simple accumulation of toxins due to food-web transfer. Based on the discovery of toxin-producing bacterium in
the cells of toxic dinoflagellates, this study suggests that PSP toxins in toxic dinoflagellates are
catabolites of bacterial substance in dinoflagellate cells. On the other hand, tetrodotoxin (TTX),
a puffer toxin, is reported to be produced by some species of bacteria. Thus, the origin of TTX of
puffer is considered to be bacteria. However, the mechanism for puffer to possess TTX through
bacteria is unknown. The present study revealed that organisms bearing either TTX or PSP toxins possess both toxins, although the proportion of both is different among the specimens. In
fact, a significant level of TTX is detected in A. tamarense. In addition, TTX-like toxin-producing bacteria are found to be infected in the liver of toxic specimens of puffer. These findings
strongly suggest that a similar mechanism between bacteria and toxic organisms is involved in
the production of TTX and/or PSP toxins in toxic organisms.
1. Introduction
Saxitoxin (STX) and its analogs (STXs) are potent
neurotoxins that block voltage-gated sodium channels on
excitable cells (Kao 1966). These toxins are produced by
several species of dinoflagellates such as Alexandrium
tamarense and A. catenella (Schantz 1986). During a
bloom of these species, filter-feeding bivalves become
toxic by ingesting them. Human consumption of toxic
shellfish results in paralytic shellfish poisoning (PSP)
owing to its main symptom, paralysis. Bivalve farming
areas infested by these species run costly monitoring programs to check for the shellfish toxicity and the causative dinoflagellates in the water. When these are present,
regular tests for toxins in associated seafood products such
© 2010 TERRAPUB, Tokyo. All rights reserved.
doi:10.5047/absm.2010.00301.0001
Received on September 8, 2009
Accepted on October 13, 2009
Published online on
April 9, 2010
Keywords
• paralytic shellfish poisoning
toxins
• saxitoxin
• gonyautoxin
• tetrodotoxin
• dinoflagellate
• Alexandrium tamarense
• bivalve
• intracellular bacteria
• bacterial infection
as bivalves are carried out and often result in the prohibition of harvesting these products. Thus, the contamination of bivalves with STXs has posed severe problems
for the bivalve culture industries as well as for public
health. Many studies have been carried out on the toxinproducing dinoflagellates and the toxins they produce and
there is much known about their taxonomy and ecology
(Steidinger 1993). The chemical structures and pharmacological properties of the toxins have been well defined
(Schantz 1986; Kao 1966). On the biosynthesis of the
toxin, Shimizu et al. (1984) proved that the skeleton of
STXs is synthesized from arginine and acetate based on
the tracer experiments using PSP-producing cyanobacterium Aphanizomenon flosaque, and proposed the unique
biosynthetic pathway. Very recently, this pathway has been
2
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 1. Structures of PSP toxins
proved to be true with slight modification through chemical and biochemical studies (Kellmann et al. 2008a, b).
However, these data are also obtained from toxinproducing cyanobacteria. Currently there is no information on the biosynthesis of PSP toxins in toxin-producing
dinoflagellates, although PSP toxins have been detected
in bacteria isolated from toxin-producing dinoflagellates
(Kodama et al. 1988; Doucette and Trick 1995; Gallacher
et al. 1997). Studies on the role of toxin-producing bacteria in the marine ecosystem are considered to be becoming more important.
2. PSP toxins found in bivalves and dinoflagellates
STX and its derivatives belong to a group of
neurotoxins which block voltage-gated sodium channels
on excitable cells (Kao 1966). These toxins are known to
be produced by several species of dinoflagellates such as
Alexandrium tamarense and A. catenella (Schantz 1986).
During a bloom of these species, bivalves, filter-feeding
mollusks, accumulate toxins by ingesting them. The PSP
toxins consist of a group of toxin components, the structures of which are similar to each other. These compounds
are water soluble alkaloids. A typical toxin is STX which
was first isolated from the toxic Alaskan butter clam
Saxidomus giganteus (Schantz et al. 1957). As STX is
highly hygroscopic and hard to crystallize, extended efforts were required to elucidate its chemical structure.
Finally, Schantz et al. (1975) succeeded in determining
its chemical structure, which possesses a characteristic
doi:10.5047/absm.2010.00301.0001
tricyclic ring system with two guanidium moieties and a
hydrated ketone. Subsequently, various derivatives of
STX, such as neoSTX and gonyautoxin (GTX) 1–4, and
B and C toxins, have been isolated from contaminated
shellfish as well as the causative dinoflagellates, and their
chemical structures have been identified (Oshima et al.
1977; Shimizu et al. 1978; Hall et al. 1980; Kobayashi
and Shimizu 1981; Shimizu and Hsu 1981; Wichmann et
al. 1981; Harada et al. 1982; Koehn et al. 1982). Currently, more than 20 derivatives are known (Oshima
1995a). The toxin in shellfish and toxic dinoflagellates is
usually a mixture of two or more toxin components.
Among them, STX is rather a minor component. Figure 1
shows the structures of the components commonly found
in toxin-contaminated shellfish and the causative
dinoflagellates. All of these are derivatives of STX. As
shown in Fig. 1, the toxins can be classified into two categories: N1-H type and N1-OH type groups. The simplest component of the former group is STX, and that of
the latter is neoSTX. Another structural variation in both
groups is the presence or absence of an O-sulfate at C11
position and an N-sulfate on the carbamoyl groups. These
variations greatly influence the physical and chemical
characters as well as the pharmacological characters.
Generally, the toxins are considered to be water soluble
and heat stable. The stability varies greatly, depending
on the pH and the structures (Shimizu 2000). At an alkaline pH, all the toxin components degrade quickly, even
at room temperature. Generally the toxins are stable under acidic conditions and it is noteworthy that the po© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
3
Table 1. Specific toxicity of each toxin component (MU·µmol–1)*.
Component
Specific toxicity
Component
Specific toxicity
STX
neoSTX
dcSTX
GTX1
GTX2
GTX3
GTX4
2483
2295
1274
2468
892
1584
1803
dcGTX2
dcGTX3
GTX5
C1
C2
C3
C4
1617
1872
160
15
239
33
143
*MU: mouse unit; one MU is a dose of toxin which kills one 20-g mouse (ddY strain) in 15 min.
According to Oshima, Y. Post-column derivatization HPLC methods for paralytic shellfish poisons. In: Hallegraeff GM,
Anderson DM, Cembella AD (eds). Manual on Harmful Marine Microalgae. IOC Manuals and Guides No. 33. Intergovernmental Oceanographic Commission, UNESCO, Paris. 1995; pp. 81–111.
tency of a toxin is different among toxin components.
Table 1 shows the specific toxicity of each toxin component expressed as mouse units (MU) per µm mole of toxin,
in which 1 MU is a dose of toxin sufficient to kill a male
mouse (ddY strain) in 15 min (Oshima 1995a).
3. Accumulation of paralytic shellfish poisoningtoxins in bivalve during a bloom of toxic
dinoflagellates in the field
PSP is a dangerous food poison because of its high
fatality, acuteness in developing symptoms, and worldwide occurrence. It is confirmed by many authors that
bivalves become toxic during blooms of toxic
dinoflagellates. PSP has long been known along the Pacific and Atlantic coasts of North America and Canada,
and many fatal cases have been recorded (Prakash et al.
1967). Poisoning occurred in September of 1972 and in
June and September of 1974 after large-scale red tides of
A. tamarense occurred along the East Coast, and 26 persons were intoxicated during the 1972 incidence. It was
prohibited to take shellfish along a 3200 km coastline,
which resulted in great economic loss (LoCicero 1975).
In Japan, PSP had not been recognized before the incident at Toyohashi in 1948 (Hashimoto et al. 1950). Since
then, a few cases of PSP have been observed and so detailed surveys on the causative dinoflagellates and bivalve
toxicity have not been conducted. Ofunato Bay is one of
the places where PSP due to consumption of scallop
Chlamys nipponensis akazara occurred in 20 people including one fatal case in 1961 (Kawabata et al. 1962).
The toxin in toxic shellfishes, as well as possibly causative organisms in the bay, has been examined by some
investigators (Murano 1975), though not satisfactorily.
After the 1977 incident, Fukuyo (1979) isolated an
Alexandrium species from the bay, and identified it as
Gonyaulax excavate (Alexandrium tamarense). Oshima
and Yasumoto (1979) showed that the toxin from cultured
A. tamarense consisted of GTX1, 2, 3, 4 and neosaxitoxin
doi:10.5047/absm.2010.00301.0001
(neoSTX). Fukuyo (1979) observed that toxic
dinoflagellates in Ofunato Bay presented three peaks in
abundance in 1979. They found that the first and second
peaks appearing in spring and summer, respectively, consisted of Alexandrium tamarense (former Protogonyaulax
tamarensis) and the third peak in autumn consisted of
A. catenella (former P. catenella).
In order to make clear any possible relationship
between the appearance of toxic dinoflagellates and the
toxification of the scallop feeding on those plankton, the
occurrence of Alexandrium spp. in Ofunato Bay was
monitored in association with the toxin level of scallop
Patinopecten yessoensis in Ofunato Bay (Ogata et al.
1982). Seawater samples for monitoring the cell density
of A. tamarense were collected, usually twice a week,
from different depths of water (2 m-intervals) at Shizu
(St. S. 24 m depth) and Yamaguchi (St. Y. 14 m depth) in
Ofunato Bay (Fig. 2). Some 500 mL of seawater was concentrated by gravity filtration using a membrane filter.
The concentrate was made up to 20 to 40 mL and each
1 mL portion was used for microscopic counting of
Alexandrium cells. When the population of Alexandrium
tended to increase, seawater samples were collected almost every day. Nontoxic scallop Patinopecten yessoensis
were transplanted from Mutsu Bay, Aomori Prefecture,
to the two stations at a 10 m depth. Five scallop specimens were collected for testing for toxicity whenever the
seawater sampling was done. Digestive glands were excised from them, combined and measured for PSP toxicity by the AOAC method (Horowitz 1984).
Alexandrium appeared twice in spring and again
in autumn in this bay. Similar results were obtained from
both stations. The results from St. S in Fig. 3 show that
relatively small number of Alexandrium began to appear
in February and March, and disappeared in April at both
stations. The plankton appeared again in May and sharply
increased its density, showing large fluctuations. It diminished by the end of June at both stations. Alexandrium
appeared again in September and reached maximum
© 2010 TERRAPUB, Tokyo. All rights reserved.
4
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 2. Map of Ofunato Bay showing two sampling station.
Reprinted with permission from Bulletin of the Japanese Society of Scientific Fisheries, 48, Ogata et al., The occurrence of
Protogonyaulax spp. in Ofunato Bay, in association with the
toxification of the scallop Patinopecten yessoensis. 563–566,
Fig. 1, © 1982, the Japanese Society of Fisheries Science.
abundance in mid-October at both stations. It disappeared
by the end of October, showing that it appeared three times
in 1980. The Alexandrium which occurred in spring and
summer was identified as A. tamarense while that occurred in autumn was identified as A. catenella. In a continuous survey in the bay over 10 years, similar patterns
were observed. Toxicity of scallop increased in association with the occurrence of Alexandrium. However, the
ratio of the toxicity level to the cell number significantly
varied, even in the same species. The ratio for the spring
A. tamarense was generally larger than for that of summer, suggesting that both organsms differed substantially
from each other in the amount of toxin per cell. It has
been reported that the amount of toxin contained in a cultured A. tamarense cell varied depending upon growth
conditions (Ogata et al. 1987a). The toxin contents of
natural cells in the field seemed to reflect their physiological conditions. Once intoxicated, scallop did not
become nontoxic in July and August, even during several months after the responsible plankton disappeared.
They still maintained considerable amounts of toxin, indicating that the toxin can not be eliminated easily from
doi:10.5047/absm.2010.00301.0001
Fig. 3. Seasonal changes in cell number of Alexandrium (—)
and in toxicity level of scallop (---) at St. Shizu of Ofunato
Bay. Reprinted with permission from Bulletin of the Japanese
Society of Scientific Fisheries, 48, Ogata et al., The occurrence
of Protogonyaulax spp. in Ofunato Bay, in association with the
toxification of the scallop Patinopecten yessoensis. 563–566,
Fig. 3, © 1982, the Japanese Society of Fisheries Science.
the scallop. As shown in Fig. 4, it has been confirmed by
the toxicity change of highly toxic scallop which was
reared in an aquarium (Oshima et al. 1982a). When the
highly toxic scallop was reared in Alexandrium-free water, around two thirds of toxin was lost within 1 week.
However, a considerable amount of toxin was maintained
for a long period. As described above, the species appearing in spring was A. tamarense while that in autumn
was A. catenella. The scallop toxicity was correlated with
the abundance of A. tamarense (Fig. 4), whereas the association of A. catenella with shellfish toxicity was not
clear in this bay.
Interestingly, some curious phenomena were observed during the monitoring. One of them is that there
was no marked toxicity increase in the shellfish when
A. tamarense reached the maximum abundance. The
toxicity of scallop increased to the maximum, then the
population of A. tamarense was decreasing. The timelag between the two maxima was about one week. Another is that the scallop showed a repeated rise and fall in
toxicity during the period, when the supply of the toxin
seemed to be apparently suppressed. These apparently
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
5
ins due to exposure to toxic bloom of dinoflagellate, become toxic because of ingestion of toxic feces from toxic
scallop (Kikuchi et al. 1996). These facts show that scallop possibly accumulate toxins from toxic feces of the
toxic scallop as well as from toxic dinoflagellates. Thus,
it seems to be difficult to understand the kinetics of toxins accumulated in scallop from dinoflagellates from the
data on field survey.
4. Toxin production of Alexandrium species
Fig. 4. Decline of the toxicities of scallop Patinopecten
yessoensis (a) and mussel Mytilus galloprovincialis (b) in laboratory tanks. Reprinted with permission from Bulletin of the
Japanese Society of Scientific Fisheries, 48, Oshima et al., Features of paralytic shellfish poison occurring in Tohoku district.
525–530, Fig. 4, © 1982, the Japanese Society of Fisheries
Science.
curious phenomena may be due to the difference of the
toxin content of the cells under different environmental
and/or physiological conditions. Accurate analysis of the
parameters such as toxin contents of the moving plankton cells is actually difficult. Thus the one week time-lag
observed between the peaks of shellfish toxicity and abundance of toxic dinoflagellate may be within the limit allowed usually in the fixed point observation on the sea.
However, similar phenomena have been observed almost
every year in the monitoring in Ofunato Bay (Sekiguchi
et al. 1989) and in Okirai Bay in the same prefecture using mussel Mytilus galloprovincialis as a monitoring bivalve (Oshima et al. 1982a).
This phenomenon is difficult to explain if the toxins in the dinoflagellates transfer to the shellfish via the
food chain. It is hard to evaluate the exchange of the toxin
amount between dinoflagellates and shellfish in a field
survey in which samples of shellfish and plankton are
collected periodically at a station set in the field, even
though the frequency of the sampling is increased. In
addition, it is pointed out that nontoxic scallop introduced
to the scallop culture area where scallop accumulated toxdoi:10.5047/absm.2010.00301.0001
Toxin production of the toxic dinoflagellate has been
indicated to be affected by a variety of physiological or
environmental factors: the toxicity of A. tamarense is different in its various growth stages (Prakash 1967; White
and Maranda 1978; Oshima and Yasumoto 1979; Singh
et al. 1982; Boyer et al. 1985); salinity affects the toxin
production of A. tamarense (White 1986); toxin production of A. tamarense changes depending on the different
growth stage of the cells (Kodama et al. 1982a). Here we
describe the effect of water temperature and light intensity on growth rate and toxicity change in A. tamarense.
A clonal culture was obtained from a single vegetative cell of A. tamarense isolated from Ofunato Bay, Iwate
Prefecture, Japan in March, 1984, when the species was
blooming. The clone obtained was mass-cultured and
maintained in enriched seawater T1 medium (Ogata et
al. 1987b) at 15°C under an illumination of 3000 lux with
an LD cycle of 16 h:8 h. This clone was cultured under
different temperatures and light intensities after
acclimation of the clonal population during a period of
20 days. An aliquot of the cells from each culture was
taken every day to count the cell density under a microscope. The growth rate (µ2 was estimated from cell counts
in the exponential phase according to Fukazawa et al.
(1980).
The growth rate decreased with the decrease of
water temperature and light intensity. The toxicity of the
cell increased with a decrease of growth rate (Fig. 5). Interestingly, this growth rate was decreased by lowering
the temperature rather than by lowering the light intensity. This fact coincided well with our observation that
the toxicity of A. tamarense at a lower temperature was
higher than that at a higher temperature (Ogata et al.
1987a). This fact suggests that photosynthesis is necessary for the production of toxin in A. tamarense. In order
to confirm the effect of light on the toxin production of
A. tamarense, we inhibited growth by lowering either
water temperature or light intensity during mid-exponential phase, and then examined the toxicity of the cells
before and after treatment. As shown in Fig. 6, growth
stopped when either temperature or light intensity was
lowered. After this treatment, toxicity of the cells (the
growth of which was slowed by lowering temperature)
increased, while that by lowering light intensity did not
(see Table 2). These facts indicate that photosynthesis is
© 2010 TERRAPUB, Tokyo. All rights reserved.
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M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 5. Alexandrium tamarense. Relation between toxicity and
growth rates measured in different water temperature (a) and
light intensity (b). Different toxicity formulas are used in A
and B. Reprinted with kind permission from Springer Science
+ Business Media: Marine Biology, Effect of water temperature and light intensity on growth rate and toxicity change in
Protogonyaulax tamarensis, 95, 1987, 217–220, Ogata,
Ishimaru and Kodama, Fig. 3.
Glover et al. (1975) showed that amino acids were produced during short periods of photo-assimilation. We
added NO3 as a nitrogen source in the culture experiments.
Therefore, de novo synthesis of amino acids should be
carried out utilizing incorporated NO3. The NO3 assimilation of microalgae is reported to be light-dependent
(Morriss 1974; Syrett 1981), and this was also confirmed
in A. tamarense (MacIsaac et al. 1979). Therefore, a decrease of light intensity at mid-exponential phase of our
experiments seems to significantly retard de novo synthesis of amino acids by suppressed nitrogen assimilation. The interruption of toxin production observed during the light retardation at the mid-exponential phase
seems to depend on suppressed de novo synthesis of amino
acids because of the decrease of NO3 assimilation. From
these, de novo synthesis of amino acids by photo-assimilation is thought to play an important role in the toxin
production of A. tamarense.
5. Transformation of toxins in dinoflagellates and
bivalves
Fig. 6. Alexandrium tamarense. Growth curves obtained by
lowering the temperature (a) and light intensity (b) in the midexponential phase. Reprinted with kind permission from
Springer Science + Business Media: Marine Biology, Effect of
water temperature and light intensity on growth rate and toxicity change in Protogonyaulax tamarensis, 95, 1987, 217–220,
Ogata, Ishimaru and Kodama, Fig. 5.
necessary for the toxin production of A. tamarense. They
also explain well the observation of the field survey that
the toxin content of A. tamarense occurred in March and
is higher than that occurred in May to June (Ogata et al.
1982).
Shimizu et al. (1984) showed that STX analogs were
synthesized from arginine and acetic acid in A. tamarense.
doi:10.5047/absm.2010.00301.0001
PSP toxins have been detected in several species of
Alexandrium, Gymnodinium catenatum and Pyrodinium
bahamense var. compressum. These species are photosynthetic dinoflagellates. Thus, PSP toxins are considered to be produced by these dinoflagellates. This view
is supported by the fact that toxin production has been
confirmed in axenic cultures of these species (Burke et
al. 1960; Singh et al. 1982). That metabolism of toxins,
including biosynthesis, occurs in the cells of these
dinoflagellates was indicated by these experiments. Various toxin components found in the toxic species of
dinoflagellates could be metabolites of toxin metabolism
that occurred in the dinoflagellates. Interestingly, the
toxin profile of cultured cells does not change significantly under different culture conditions such as different temperatures, light intensities, and levels of nutrients,
if the culture is monoclonal (Boyer et al. 1986, 1987;
Cembella et al. 1987; Ogata et al. 1987b). Therefore, the
toxin profiles of dinoflagellates isolated from various areas have been elucidated by many research groups. These
studies indicate that the toxin profile is often specific to a
species of dinoflagellates. For example, G. catenatum
shows a characteristic toxin profile which consists of
N-sulfocarbamoyl toxins such as B and C toxins as major components (Oshima et al. 1990). GTXs are rarely
detected in the species. On the other hand, P. bahamense
var. compressum contains B toxins and their carbamate
counterparts, STX and neoSTX, as dominant toxins. A
significant amount of decarbamoyl saxitoxin (dcSTX)
(see the structure in Fig. 1) is also found in this species
(Harada et al. 1982, 1983). A difference in toxin profile
has also been observed among species belonging to the
genus Alexandrium. C2 and GTX4 are the dominant
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
7
Table 2. Alexandrium tamarense (OF84423D3). Effect of slowdown of the growth rate on toxicity.
Treatment
Harvesting
Toxicity
Total toxin concentrationa
MU/104 cell
MU/mm3
(nmol/104 cells)
Ab
Before treatment
After treatment
0.68
1.63
2.30
4.84
0.491
1.007
Bc
Before treatment
After treatment
0.64
0.68
2.71
3.13
0.398
0.494
Calculated from the toxin composition
Treatment A: Growth rate was slowed by lowering temperature at the mid-exponential phase
c
Treatment B: Growth rate was slowed by lowering light intensity at the mid-exponential phase
Reproduced with kind permission from Springer Science + Business Media: Marine Biology, Effect of water temperature and
light intensity on growth rate and toxicity change in Protogonyaulax tamarensis, 95, 1987, 217–220, Ogata et al., Table 3.
a
b
components in Alexandrium (Protogonyaulax) tamarense
and A. catenella (Oshima et al. 1990), while GTX1–4
are dominantly found in A. tamiyavanichii (Kodama et
al. 1988; Ogata et al. 1990). This species was isolated
from the Gulf of Thailand and identified as A. cohorticula,
but it was reclassified as a new species A. tamiyavanichii
by Balech: A. ostenfeldii showed a toxin profile consisting of B toxins and their carbamate counterparts as dominant toxins (Hansen et al. 1992).
Strains of the same species isolated from different
geographical areas often show the different toxin profile.
Oshima et al. (1993a) showed that a Tasmanian isolate of G. catenatum contains C4, which was absent in
the same species isolated from other areas (Ikeda et
al. 1989). Furthermore, they detected novel components, 13-deoxydecarbamoyl derivatives, in some strains
of G. catenatum (Oshima et al. 1993b). A similar phenomenon was also observed among different strains of
A. minutum. Most of the isolate of A. minutum from various areas show a characteristic toxin profile consisting
of GTX1–4 (Oshima et al. 1989; Franco et al. 1994). In
contrast, neoSTX, STX, and C toxins were detected in an
isolate from New Zealand (Chang et al. 1997). Another,
similar phenomenon was observed among clones of
A. tamarense (=Protogonyaulax tamarensis) isolated
from different areas of Japan (Oshima et al. 1982b).
It is noteworthy that nontoxic strains are often
found in toxic species. Some strains of A. tamarense
are reported to be nontoxic (Loeblich and Loeblich III
1975). Oshima et al. (1993b) also reported that a clonal
culture of G. catenatum established from a cyst isolated
from Tasmania is nontoxic. These facts may indicate that
PSP toxins are not essential for the survival of these
dinoflagellates. Although little is known about the metabolism of toxins in dinoflagellates, some enzymes involved in toxin transformation have been reported.
Oshima (1995b) showed the presence of oxidase activity
doi:10.5047/absm.2010.00301.0001
in some clones of A. tamarense that catalyzes the oxidation of N1-H of GTX2,3 to form GTX1,4. However,
this enzyme is not always present in all the isolates of
A. tamarense. On the other hand, N-sulfotransferase that
converts carbamate toxins to the corresponding C toxins
has been detected in G. catenatum (Oshima 1995b;
Yoshida et al. 1996). The occurrence of these enzymes
may explain the characteristic toxin profile of some
dinoflagellate species. The hereditary character of the
toxin profile in dinoflagellates supports the role of enzymes in the characteristic toxin profile of dinoflagellates.
Various toxin components found in the dinoflagellates
may be metabolites derived from a de novo product in
the biosynthesis of the STX skeleton, though so far this
is not known with certainty. It is plausible that enzymes
are involved in the transformation of the de novo toxin to
various toxin components.
The profile of the toxins accumulated in shellfish
during a bloom of toxic dinoflagellates reflects that of
the causative dinoflagellate. However, the toxin components accumulated in the shellfish change after the causative dinoflagellates disappear from the environment
(Oshima 1995a). The shellfish themselves are not involved in biosynthesis of the toxins, and thus the toxin
components transferred from the dinoflagellates undergo
metabolic transformation in the shellfish. In an earlier
study, Shimizu and Yoshioka (1981) showed that transformation of PSP toxins occurs in the shellfish. They incubated an homogenate of the scallop Patinopecten
magellanicus contaminated with neoSTX and GTX2,3,
and found that the proportion of STX increased after incubation. These facts showed that neoSTX and GTXs are
converted to STX in the shellfish. From these data, they
suggested the presence of enzymes in the shellfish which
are involved in the transformation of toxin components,
though little is known about such enzymes. The rate of
the transformation is very low indicating that enzyme
© 2010 TERRAPUB, Tokyo. All rights reserved.
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M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
activity in the scallop is low, if the reaction is enzymatic.
Oshima (1995b) screened the enzyme activities which
transform C toxins in the shellfish, and detected the activity to hydrolyze N-sulfocarbamoyl of C toxins to form
dcGTXs in two species of clams, Mactra chinensis and
Peronidia venulosa. Enzyme activity to hydrolyze carbamate toxins was also detected in some species of shellfish (Sullivan et al. 1983). However, the enzyme involved
in the transformation of GTXs to STX has not been found
in shellfish, though this transformation is observed generally in the shellfish.
6. Non-enzymatic transformation of PSP toxins: formation of unique intermediate conjugates of PSP
toxins and thiols
As described above, GTXs accumulated in shellfish are gradually transformed to STXs. The rate of transformation is very slow (Oshima 1995a), suggesting that
the reaction is not enzymatic. On the other hand, Kotaki
et al. (1985) reported that a part of GTXs incubated with
bacterial cells are were transformed to STXs, showing
that these bacteria possess an ability to transform GTXs
to STXs. They also indicated that the factors involved in
toxin transformation occur widely in the ecosystem. Thus,
we incubated GTXs in the water-soluble extract of a bacterium Moraxella sp. (PTB-1) (Sakamoto et al. 2000) and
found that the extract prepared with 0.1 M phosphate
buffer (pH 7.4) often converted GTXs to STXs. However, this transformation activity was not always observed,
indicating that the activity of the extract is not stable.
However, once the activity was obtained from the bacterium, it is very stable, even after heat treatment. These
facts indicate that the activity is not due to the enzyme.
On the other hand, Asakawa et al. (1987) reported that
glutathinone (GSH), a biological reductant widely occurring in organisms, transform GTX1, 2, 3 to STX. Bacteria are known to possess a membrane-bound enzyme that
degradates GSH (Nakayama et al. 1984). If the activity
is due to GSH, a part of GSH seems to be degradated by
the enzyme during preparation of water-soluble extract
of the bacterium. Thus, the GSH levels in the extracts
with and without L -serine, the inhibitor of GSH
degradating enzyme, were analyzed. At the same time,
transformation of GTXs to STXs was also analyzed
(Sakamoto et al. 2000).
Figure 7 shows the change of GSH level in the extracts prepared from the homogenates with and without
the inhibitor. The GSH level in the extract in the absence
of the inhibitor was 0.21 mM when the extract was prepared just after the cells were homogenated (Fig. 7, upper). However, this level decreased gradually when the
homogenate was incubated at 30°C. After 24 h incubation, the level was decreased to 0.06 mM. In contrast,
GSH levels in the extract in the presence of the inhibitor
were 0.31 mM when the extract was prepared just after
doi:10.5047/absm.2010.00301.0001
Fig. 7. Change of toxin components and glutathione (GSH) in
the mixture of gonyautoxins (GTX) 2,3 and bacterial
homogenates during incubation. GTX2,3 (10 µmol·L–1) was
incubated with the homogenates at 30°C. Change of toxin
components in the homogenate with 0.1 M phosphate buffer,
pH 7.4. (b) Change of toxin components in the homogenate
with 0.1 M borate buffer, pH 7.4 containing 100 mM L -serine. 䊊, GTX2,3; 䊉, saxitoxin; white bar, GSH level. PSP, paralytic shellfish toxins. Reprinted from Sakamoto et al., Formation of intermediate conjugates in the reductive transformation
of gonyautoxins to saxitoxins by thiol compounds. Reprinted
with permission from Fisheries Science, 66, Oshima et al.,
Features of paralytic shellfish poison occurring in Tohoku district. 136–141, Fig. 2, © 2000, the Japanese Society of Fisheries Science.
the cells were homogenized (Fig. 7, lower). This level
also decreased slightly, but 0.21 mM of GSH remained
in the extract, even when the homogenate was incubated
for 24 h. These results show that GSH in bacterial cells is
easily decomposed by the GSH degradating enzyme during extraction and incubation, unless the extract was prepared in the presence of the inhibitor against the enzyme
(Sakamoto et al. 2000).
Figure 7 also shows the change of GTX2,3 added
to these homogenates during incubation. When GTX2,3
was incubated with the cell homogenate in the presence
of the inhibitor, more than 80% of GTX2,3 disappeared
within 0.5 h, although no STX was detected (Fig. 7,
lower). After 1 h incubation, most of GTX2,3 disappeared
and a trace amount of STX appeared. The level of STX
increased gradually during further incubation and reached
2.2 mM after 24 h incubation, showing that about 20% of
GTX2,3 added to the homogenate was transformed to
STX. This phenomenon also suggests the formation of
an intermediate compound which cannot be detected by
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
HPLC in the transformation reaction of GTX2,3 to STX
(Sakamoto et al. 2000).
When GTX2,3 was added to the homogenate without the inhibitor, about 20% of toxins also disappeared
within 0.5 h (Fig. 7, upper). However, the level of GTX2,3
did not change during further incubation. No STX nor
other known PSP toxin components were detected during incubation. These results show that the bacterial activity to transform GTX2,3 to STX is mainly due to GSH.
Unstable activity of bacterial extract prepared in the absence of the inhibitor seems to be caused by degradation
of GSH by the GSH degradating enzyme in the bacterial
homogenate during extraction. Membrane-bound protein
fraction prepared by 2-mercaptoethanol (ME) containing a buffer also showed a significant activity to transform GTX2,3 to STX. This activity was stable under heating and protease treatment, showing that it is not an
enzymatic reaction. The activity was also observed in the
extraction buffer, suggesting that it is the effect of 2-ME
contained in the buffer.
Figure 8 shows the change of toxin components in
the mixture of GTX1,4 and GSH during incubation at
70°C in 0.1 M phosphate buffer (pH 7.4). GTX1,4 decreased markedly and disappeared from the mixture
within 2 h. In contrast, neoSTX appeared at 30 min and
gradually increased during incubation for 2 h to 35% of
the amount of GTX1,4 added to the reaction mixture.
GTX1,4 in the phosphate buffer decreased slightly after
12 h, but no neoSTX was detected by HPLC. Similar results were obtained when GTX2,3 was incubated with
GSH. In this case, however, STX was formed instead of
neoSTX.
Figure 9 shows the change of toxin components in
the reaction mixture of GTX1,4 and 2-ME during incubation at 70°C in the phosphate buffer (Sakamoto et al.
2000). In this case, most of GTX1,4 disappeared within
2 h while a small amount of neoSTX appeared. The
amount of neoSTX increased gradually by 15% of the
original amount of GTX1,4. Similar results were obtained
from the reaction mixture of GTX2,3 and 2-ME except
that STX was formed instead of neoSTX.
These results show that thiol compounds transform
GTXs to STXs. However, the sum of GTXs and STXs in
the reaction mixtures was less than half of the original
amount of GTXs, showing that more than half of GTXs
added to the reaction mixtures disappeared. This suggests
that the conjugates of GTXs and thiol compounds are
formed in the course of the transformation. Thus, the 2 h
incubation mixtures of GTXs and GSH in which GTXs
disappeared, were heated in 1 M 2-ME at 100°C for 10
min. HPLC analysis showed that almost 100% of the
added GTXs was recovered as STXs, indicating that
GTXs and thiol compounds form conjugates and that additive molecules of thiol compounds are necessary to release STX from the conjugates.
doi:10.5047/absm.2010.00301.0001
9
Fig. 8. Change of toxin components in the mixture of
gonyautoxins (GTX) 1,4 and glutathione (GSH) during incubation. GTX1,4 (4 µM) was incubated with 1 mL of 8 mM
GSH in 0.1 M phosphate buffer, pH 7.4, at 70°C. 䊐, GTX1,4
in the reaction mixture; 䊏, neoSTX in the reaction mixture;
䊊, GTX1,4 in the phosphate buffer as a control. Reprinted with
permission from Fisheries Science, 66, Sakamoto et al., Formation of intermediate conjugates in the reductive transformation of gonyautoxins to saxitoxins by thiol compounds. 136–
141, Fig. 3, © 2000, the Japanese Society of Fisheries Science.
Fig. 9. Change of toxin components in the mixture of
gonyautoxins (GTX) 1,4 and 2-mercaptoethanol (2-ME) during incubation. GTX1,4 (4 µM) was incubated with 1 mL of
8 mM 2-ME in 0.1 M phosphate buffer, pH 7.4, at 70°C.
䊐, GTX1,4 in the reaction mixture; 䊏, neoSTX in the reaction
mixture; 䊊, GTX1,4 in the phosphate buffer as a control. Reprinted with permission from Fisheries Science, 66, Sakamoto
et al., Formation of intermediate conjugates in the reductive
transformation of gonyautoxins to saxitoxins by thiol compounds. 136–141, Fig. 4, © 2000, the Japanese Society of Fisheries Science.
Table 2 summarizes the results of TLC of the
reaction mixture of GTXs and GSH before and after
incubation. After incubation, spots of GTXs disappeared
and those of STXs appeared instead. In addition to these,
© 2010 TERRAPUB, Tokyo. All rights reserved.
10
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
spots different from those of toxin components and GSH
appeared at Rf 0.10 in the mixture of GTX1,4 and GSH
and 0.04 in the mixture of GTX2,3 and GSH, respectively,
indicating the formation of the conjugates of GTXs and
GSH. A spot corresponding to GSH became very faint
after incubation, suggesting that excess amounts of GSH
were oxidized to GSSG during incubation which could
not be detected by this system (Sakamoto et al. 2000).
In a trial to purify the conjugate by chromatography on Bio-Gel P-2, it was isolated in a pure form (Sato
et al. 2000). The high resolution FABMS and
NMR(HMBC) spectrum of the purified substances
showed that the compounds are conjugates of neoSTX
and thiols in which the C11 atom of neoSTX is covalently
bound with a sulfur atom of thiols (Fig. 10). When
GTX2,3 was incubated with thiols, the corresponding
conjugates of STX and thiols were obtained (Sato et al.
2000a).
There has been no report on the reductive elimination of sulfate ester by thiols. Thus, the transformation of
GTXs to STXs via the intermediate conjugates is considered to be a quite unique reaction. Formation of the conjugates does not occur under acidic conditions. Furthermore, it was confirmed that GTXs-12-ol reported in toxinproducing cyanobacterium (Onodera et al. 1997) and a
dinoflagellate species from tropical area (Lim et al. 2007),
analogues in which the gem diol at C12 is reduced, did
not react with thiols at all. These facts indicate that the
characterstic keto-gem diol structure at C12 of the toxins
is essential for the reaction. When the equilibrium mixture of α- and β-O-sulfate toxins is incubated with
thiols, the α-O-sulfate toxin is consumed faster than
the β-O-sulfate toxin in the formation of the conjugates,
showing that α-O-sulfate toxin is a “reactive isomer”
(Fig. 11).
Treatment of the conjugates with an excess amount
of thiols results in the formation of disulfides and STX or
neoSTX, indicating that the transformation of GTXs to
STXs consists of a two-step reaction (Fig. 11). In the first
step, the sulfur atom of the thiols attacks the electrophilic
C12 of GTXs to form a thiohemiketal, followed by the
formation of stable thioether conjugates by a 1,2 shift.
This is an intramolecular reaction in which GTXs easily
react with a low concentration of thiols at a low temperature. The second step is an intermolecular reaction between the conjugates and thiols. Therefore, it requires
more energy than the first-step reaction. In order to complete the second-step reaction within a short period, heating with an excess amount of thiols is necessary. Actually, the conjugates are formed, even in a solution of GTXs
in a low concentration of thiols, such as 1 mM or lower at
room temperature. However, only a trace amount of STXs
is obtained under this condition. In contrast, almost 100%
of STXs in the conjugates can be recovered when conjugates dissolved in 1 M or higher concentrations of thiols
are heated for several minutes.
Reduction of N1-OH type toxins to N1-H type toxins was not observed in the reaction of GTXs and thiols.
This fact does not coincide with the results of Asakawa
et al. (1987) in which GSH transformed GTX1 to STX.
In their experiments, they used partially purified toxins
containing GTX2,3 with a trace amount of GTX1. The
reaction mixtures were analyzed by electrophoresis. Probably, most of GTXs in the reaction mixture formed conjugates with GSH and disappeared from the mixture. The
amount of neoSTX derived from the conjugate was too
low to be detected by electrophoresis.
Discovery of stable 11-thiol derivatives of STX is
particularly important in terms of the metabolism of PSP
toxins. These findings show that STX derivatives are more
reactive than previously have been considered. The reaction between GTXs and thiols is useful for the development of biochemical probes such as antibody against
PSP toxins which would facilitate the study of more detailed mechanisms of the reactions in which the toxins
are involved.
Fig. 10. Structure of the intermediates in the transformation of GTX1,4 into neoSTX. Left: gluthathione-neoSTX adduct;
right: 2-mercaptoethanol-neoXTX adduct. Reprinted from Bioorganic & Medical Chemistry Letters, 10, Sato, Sakai and
Kodama, Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols,
1787–1789, © 2000, with permission from Elsevier.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
11
Fig. 11. Mechanism of reductive transformation of GTX1,4 mixture to neoSTX by thiol. Reprinted from Bioorganic &
Medical Chemistry Letters, 10, Sato, Sakai and Kodama, Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols, 1787–1789, © 2000, with permission from Elsevier.
7. Accumulation and depuration kinetics of PSP toxins in the scallop fed A. tamarense
As described above, observation of the field survey
on shellfish toxicity in association with the abundance of
causative dinoflagellates suggests that the mechanism by
which bivalves accumulate PSP toxins is more complex
than those which have been considered based on the food
web transfer theory. However, it is almost impossible to
understand how the shellfish accumulate and depurate
PSP toxins during a bloom of dinoflagellates from the
field data. Therefore, we examined this problem by the
kinetics of toxin accumulation in shellfish based on the
feeding experiments of dinoflagellate to the shellfish.
In a trial, interspecific difference in the ability to
accumulate PSP toxins were examined by rearing five
species of bivalves, scallop Patinopecten yessoensis, oyster Crassostrea gigas, mussel Mytilus galloprovincialis,
short-necked clam Ruditapes philippinarum, and ascidian
Halocynthia roretzi (Sekiguchi et al. 2001a). The specimens of each species were reared in separate tanks with
500 L of seawater and fed a known number of cultured
A. tamarense cells. After 24 h, the number of A. tamarense
cells in the tank was counted to determine the number of
doi:10.5047/absm.2010.00301.0001
ingested cells. At the same time, specimens of each animal were transferred to an aquarium with freshly prepared filtered seawater, to which was added the culture
of A. tamarense. The shellfish were then reared in the
same way. Specimens of each animal were thus fed on
A. tamarense once a day for eight days. After feeding
was completed, the specimens were further reared in the
same way without feeding for 12 days. During rearing,
three specimens of each animal were sampled every two
days for the analysis of the toxins by HPLC.
All the animals ingested most of the cells fed within
24 h. More than 90% of toxin components of the cells
consisted of C1,2 and GTX1,4, and this profile was almost constant throughout the feeding period. Figure 12
shows the change of toxin contents accumulated in each
shellfish specimen in a molar base. The amount of each
toxin component is also shown in each bar. Average values of accumulated toxins and those of incorporated toxin
amounts in a specimen calculated from the toxin contents and ingested cell number of A. tamarense, are also
shown in the figure.
All the specimens became toxic, but the toxin level
of the specimens showed remarkable individual variation,
even among the specimens sampled on the same day. To
© 2010 TERRAPUB, Tokyo. All rights reserved.
12
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 12. Toxin accumulation in four species of bivalves and an ascidian fed on cultured cells of Alexandrium tamarense.
(a) Scallop, (b) mussel, (c) oyster, (d) short-necked clam and (e) ascidian. Reprinted with permission from Fisheries Science,
67, Sekiguchi et al., Accumulation of paralytic shellfish poisoning toxins in bivalves and an ascidian fed on Alexandrium
tamarense cells, 301–305, Fig. 1, © 2001, the Japanese Society of Fisheries Science.
the contrary, no significant difference was observed in
toxin composition of the specimens sampled on the same
day. Toxin composition of each animal closely reflected
that of A. tamarense, that is, more than 90% of accumulated toxins consisted of C1,2 and GTX1,4. The proportion of C1,2 to GTX1,4 was almost constant during the
feeding experiment. These results indicate that there is
no difference in the deterioration rate among the toxin
components, although C1,2 are reported to be rapidly
eliminated over five days in the scallop Patinopecten
yessoensis exposed to a natural bloom of A. catenella in
which C2 was dominant (Oshima et al. 1990). The difference in the results between feeding experiments and
the field study may be due to the difference between natural and laboratory conditions.
After the feeding had been stopped, amounts of
STXs (STX, neoSTX and decarbamoyl-STX) increased
slightly. This trend was observed when the specimens
were reared for a longer period. These changes in toxin
profile were similar to those observed by Oshima et al.
(1990). Although a marked individual difference was
observed in the accumulated toxin levels of the three
doi:10.5047/absm.2010.00301.0001
specimens sampled at the same stage of the experiment,
the average toxin levels showed a trend toward increasing during the feeding experiments. No significant difference was observed in the maximum toxin levels among
the mussel, the short-necked clam, and the oyster which
were fed on almost equal amounts of A. tamarense
(Fig. 12). However, the ascidian accumulated about
twice more toxins than other bivalves, although they
ingested equal amounts of A. tamarense. Although
scallop were fed on three time more than dinoflagellate,
they accumulated nearly the same level of toxins as the
ascidian.
After the feeding had been stopped, toxin levels of
each species decreased. However, a considerable high
level was often observed in the scallop and the ascidian,
even after the cessation of feeding. These results show
that accumulated toxins in these animals are hardly eliminated. This observation coincided well with our previous
results that scallops maintained a high toxin level for a
long time after the disappearance of the causative
dinoflagellate in nature.
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Many studies have been carried out to discover the
kinetics of toxin accumulation of bivalves (Bricelj et al.
1990, 1991, 1996; Wisessang et al. 1991). In these studies, many shellfish specimens are kept in the same tank
and fed on cultured cells of toxic dinoflagellates, and the
shellfish toxicity was analyzed by using a homogenate of
several specimens to minimize the error of individual differences. The current study unexpectedly revealed that
bivalves showed considerable individual variations in
toxicity. These differences are considered to be caused
by different feeding behaviors among the specimens,
probably due to the difference in feeding behavior owing
to the sensitivity of handling or a sudden change of environment among the specimens. Thus, the feeding experiments should be designed to take feeding behavior of
shellfish into consideration.
As handling of the shellfish during the experiments
was found to largely affect the feeding behavior of the
shellfish (Sekiguchi et al. 2001a), a single specimen of
shellfish was reared in a single tank, and the known
amount of cultured cells of dinoflagellate was fed to the
shellfish specimen (Sekiguchi et al. 2001b). Shellfish
accumulate toxins by ingesting the toxic dinoflagellate.
Occurrence of toxins in rearing water showed that a part
of the toxins was released to the environmental water,
even while ingesting the dinoflagellate. When the feeding stopped, the shellfish continuously excreted the toxins. The profile of excreted toxins was similar to that accumulated in the shellfish, indicating that shellfish release the toxin components non-selectively.
Interestingly, the amount of toxins accumulated in
the shellfish was not parallel to that of the dinoflagellate
cells fed to the shellfish (Fig. 13). At the earlier period of
the experiment when shellfish were ingesting the cells,
the amount of toxins accumulated in the shellfish was
more than the supplied toxins from dinoflagellates fed to
the shellfish. In the later period when the feeding was
stopped, the sum of the toxins in the shellfish and rearing
water had decreased to a level which was less than that
introduced from the dinoflagellates cells, showing that
some of the toxins disappeared from the experimental
system. However, this level recovered to almost the same
level as that derived from the fed cells of the
dinoflagellate, when they were further reared. These facts
indicate that a part of the toxins was transformed to an
unknown form in the shellfish which could not be detected by chemical analysis such as HPLC applied in our
study. The unknown form of the toxins is gradually transformed to toxins again in the shellfish which can be detected by chemical analysis. The unexpected high level
of toxins accumulated in the shellfish, more than the fed
amount of toxins, may show that an unknown form also
occurs in the dinoflagellate fed to the shellfish. Thiol compounds of biological origin, such as GSH, are thought to
be involved in the appearance and disappearance of the
toxins in shellfish and dinoflagellate. Proteinous thiols
doi:10.5047/absm.2010.00301.0001
13
Fig. 13. Change in amounts of PSP toxins accumulated in scallop specimens reared separately in individual tanks. The integrated amounts of toxins released from a single specimen and
those supplied to each specimen by feeding Alexandrium
tamarense cells are also shown. The sample was accidentally
lost during analysis. Reprinted with permission from Marine
Ecology Progress Series, 220, Sekiguchi et al., Accumulation
and depuration kinetics of paralytic shellfish toxins in the scallop Patinopecten yessoensis fed Alexandrium tamarense, 213–
218, Fig. 3, © 2001, Inter-Research.
such as those with a cysteine residue are possibly involved
in the formation of the conjugates with the toxins. These
thiol-bound toxins possibly react with other thiols in the
shellfish and dinoflagellates to release toxins. Chemical
transformation of the toxins observed in in vitro experiments could be involved in the transformation in
dinoflagellates as well as in shellfish. These findings suggest that PSP toxins undergo a complex metabolism in
shellfish as well as in dinoflagellates.
8. Change of toxin productivity in Alexandrium cells
There are various data on the toxicity of different
strains of A. tamarense isolated from various regions of
the world (White and Maranda 1978; Oshima and
Yasumoto 1979; Kodama et al. 1982a; Singh et al. 1982;
Boyer et al. 1985; Ogata et al. 1987a). As the toxicity of
A. tamarense is affected by physiological or environmental factors, these data cannot be directly compared. However, the toxicities of these strains seem to differ from
each other. White (1986) reported the high toxin content
of A. tamarense in nature. He also demonstrated that the
toxicity of the cell in nature fluctuated during the bloom,
suggesting that the toxicity of the cells is different in different water masses, even if they bloom in the same area
during the same period. Alternatively, these data suggest
that the toxicity differs in different clones from one another. Therefore, we examined the toxicity of various
clones isolated from the same area during the same season, by culturing them under the same conditions (Ogata
© 2010 TERRAPUB, Tokyo. All rights reserved.
14
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 14. Toxicity of various clones of Alexandrium tamarense
as a function of growth rate. These clones were grown at 15°C
under 3000 lux in medium T1 (Ogata et al. 1987b). Toxicity is
expressed in mouse unit (MU) according to Horwitz (1984)
where 1 MU represents the dose to kill a 20 g male mouse within
15 min. The growth rate (µ2) is estimated from cell counts during the exponential phase according to the method of Fukazawa
et al. (1980). Reprinted from Toxicon, 25, Ogata, Kodama and
Ishimaru, Toxin production in the dinoflagellate Protogonyaulax
tamarensis, 923–928, © 1987, Pergamon Journal Ltd. with permission from Elsevier.
et al. 1987a). Figure 14 shows the toxicity plotted
against their growth rate ( µ 2 ) for various clones of
A. tamarense which were grown under the same conditions. The data points are randomly scattered, showing
that the growth characteristics and toxin production were
different, depending on the clone. The maximum differences in toxicity and growth rate were about 100- and 4fold, respectively. HPLC-analysis of the toxin components
revealed that these clones had toxin profiles similar to
each other. These results indicate the occurrence of various clones of A. tamarense which possess different growth
characteristic and toxin productivity. This can be explained by the heterothallism of A. tamarense (Turpin
et al. 1978; Anderson 1980; Yoshimatsu 1992). According to this theory, toxin productivity as well as the growth
rate of A. tamarense could be inheritance.
In order to know the heredity of the toxin productivity of A. tamarense, we established 20 subclones
from a single clone isolated from Ofunato Bay. These
subclones were grown under the same conditions, as
described above, and the toxicity and growth rate were
analyzed. In Fig. 15, the toxicities of the subclones are
plotted against their growth rate. Most of the subclones
showed a similar growth rate. In contrast, their toxicity
varied significantly. The maximum difference in toxicity
of the subclones was about 20 fold. These results suggest
that the toxin production of A. tamarense is not a heredidoi:10.5047/absm.2010.00301.0001
Fig. 15. Toxicity of subclones from a single clone of
Alexandrium tamarense (OF84423D-3) as a function of growth
rate. These subclones were grown at 15°C under 3000 lux in
medium T1. The toxicity (MU) and the growth rate (µ2) are as
defined as in Fig. 14. Reprinted from Toxicon, 25, Ogata,
Kodama and Ishimaru, Toxin production in the dinoflagellate
Protogonyaulax tamarensis, 923–928, © 1987, Pergamon Journal Ltd. with permission from Elsevier.
tary character but an acquired one, unless mutation occurred during the culture experiments. In other words,
the information on toxin production is not coded in the
genes of A. tamarense.
These experiments were carried out within one year
after the isolation of the clone from which subclones were
prepared. Formation of the zygote cannot occur during
culture because of the heterothallism of A. tamarense
(Turpin et al. 1978; Anderson 1980; Yoshimatsu 1992),
indicating that the genetic type of each subclone was identical during our experiments. If mutation occurred, there
should have been some groups of clones with different
characteristics. However, the toxicities of subclones were
different from each other, suggesting that the variation in
the toxicity among the subclones is unlikely because of
mutation.
In conclusion, these results suggest that toxin production of A. tamarense is not a hereditary characteristic, i.e. the information on toxin production is not
coded in the genes of A. tamarense. This idea means
that toxin production in A. tamarense is an acquired characteristic.
Recently, two groups reported separately the same
results on nontoxic subclones obtained from a toxic single clone of Alexandrium. Martins et al. (2004) described
that possible explanations for the change include genetic
mutations or the effects of prolonged treatment of the toxic
culture with antibiotics. Omura et al. (2003) describe that
they prepared many subclones from non-axenic parent
culture of A. tamarense obtained from a single cell
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
15
collected from Ofunato Bay in the same way as described
above. Among these subclones, about half were non-toxic.
They proposed that this ratio is too large to explain that
loss of toxin production in subclones is caused by mutation. Some external factors may be involved in the toxin
productivity of toxic dinoflagellates. No significant difference was observed in growth and other physiological
characteristics between toxic and non-toxic clones. These
facts suggest that PSP toxins are not essential for the survival of these dinoflagellates, but rather are foreign substances for them.
9. Bacterial production of saxitoxin
It has been described above that a large difference
is observed in the toxicity among various subclones obtained from a single clone. This finding strongly suggests
that toxin production of A. tamarense is not a hereditary
characteristic. Bacterial symbiosis or infection is commonly observed in higher plants. Thus, it is plausible
that the bacteria are involved in toxin production of
A. tamarense.
A strongly toxic clone of A. tamarense was treated
with a mixture of antibiotics to obtain the axenic culture.
The culture was tested for the presence of bacteria and
confirmed that the culture medium was bacterium-free.
A. tamarense cells were harvested from thus-obtained bacteria-free culture, washed several times with sterilized
seawater, lightly homogenized, and then inoculated onto
agar plates of a nutrient medium. After incubation for
several days, bacterial colonies were observed on the
plates. The obtained bacterial strain was mass-cultured
and bacterial cells were extracted for toxin analysis. As
the toxicity was detected by mouse bioassay, the extract
containing crude toxin was prepared from more than
70 L of culture. The toxic substance was isolated by using several types of chromatographies. The purified substance was indistinguishable from authentic STX in the
analysis by different types of chromatographies such as
TLC, electrophoresis, and HPLC for PSP toxins (Kodama
et al. 1988). Typical results from HPLC analysis are
shown in Fig. 16. The peak height in HPLC to the mouse
toxicity ratio also coincided well with that of standard
STX, showing that the obtained bacterium produces STX.
This toxigenic bacterium (PTB-1) was tentatively identified as “Moraxella sp.” [later classified as a Pseudomonas/
Alteromonas species (Doucette and Trick 1995)] and most
recently as a new genus within the α-proteobacteria
(Kopp et al. 1997). Doucette and Trick (1995) and
Franca et al. (1995) employed HPLC methods to examine toxin production characteristics of Moraxella sp. and
another bacterium obtained from the toxic dinoflagellate
A. lusitanicum. Both groups reported PSP toxins in bacterial cell extracts. Gallacher and co-workers have made
an important step in confirming the synthesis of PSP toxins by bacteria isolated from five Alexandrium cultures,
doi:10.5047/absm.2010.00301.0001
Fig. 16. Identification of the bacterial toxin as saxitoxin by
HPLC-fluorometric analysis. A, saxitoxin standard (0.2 MU);
B, bacterial toxin (0.3 MU). Reprinted from Agricultural and
Biological Chemistry, 52, Kodama et al., Bacterial production
of saxitoxin, 1075–1077, © 1988, the Agricultural Chemical
Society.
including the A. lusitanicum strain noted above (Gallacher
et al. 1997), using the capillary electrophoresis-mass
spectrometry (CE-MS) method.
On the other hand, Silva (1979) presented the hypothesis that dinoflagellate toxicity is due to intracellular
bacteria. We also suggested that toxin production by
A. tamarense is not of hereditary characteristics (Ogata
et al. 1987b). The finding of toxin-producing bacterium
in the cells of A. tamarense support both Silva’s hypothesis and our previous results. The toxin production by
our bacterium was considerably lower than that by
A. tamarense, suggesting that symbiosis or parasitism
with A. tamarense enhances the toxin production by this
bacterium. In addition, only STX could be detected in
Moraxella sp., whereas the main toxin components of
A. tamarense are GTXs (Ogata et al. 1987a, b). Thus, the
toxin production of Moraxella sp. under various condition was surveyed. As shown in Fig. 17, Moraxella sp. in
shaking culture grew faster than those in standing or rotatory culture at 25 and 37°C. Figure 18 shows time course
of toxicity in the cell extract of Moraxella sp. grown under various culture conditions. No significant toxicity
was observed in any culture during 64 h. At 25°C, it
increased during 24 h, whereas it decreased after 24 h.
The cells shaken in Tryptic-Soy Broth (TSB; BBL Co.
Ltd. Tokyo) at 25°C also showed weak toxicity at 64 h
which disappeared after 168 h incubation. On the other
hand, Moraxella sp. showed considerable toxicity in all
the culture conditions when cultured in Marine Broth
(MB; Difco) at 25°C for 168 h.
Inoculates of Moraxella sp. in 1% MB and seawater
became slightly turbid after 10 days incubation, showing
that the bacterium grew only slightly in these media.
Figure 19 illustrates HPLC chromatograms of the extract
of Moraxella sp. grown in the seawater for 10 days. Peaks
© 2010 TERRAPUB, Tokyo. All rights reserved.
16
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 17. Growth of Moraxella sp. Under various culture conditions. Moraxella sp. Was cultured in Heart Infusion (䊊), Brain
Heart Infusion (䊉), Tryptic-Soy Broth (䉭), CAYE medium containing 1% Casamic acid and 1% yeast extracts (䉱), and
Marine Broth (䊏) at 25 and 37°C by standing, shaking and rotatory culture methods. Upper, 25°C, Lower 37°C; A, standing
culture; B, shaking culture; C, rotatory culture. Reprinted from Toxicon 28, Kodama et al., Production of paralytic shellfish
toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis. 707–714, © 1990, Pergamon Press plc. with
permission from Elsevier.
Fig. 18. Time course of toxin production by Moraxella sp. under various conditions. The toxicity of cells grown under
various conditions (refer to Fig. 17) was assayed by mouse neuroblast cell culture method (Kogure et al. 1988; Sato et al.
1988). Upper, 25°C; Lower, 37°C; A, standing culture; B, shaking culture; C, rotatory culture. Reprinted from Toxicon 28,
Kodama et al., Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis.
707–714, © 1990, Pergamon Press plc. with permission from Elsevier.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 19. HPLC-fluorometric analysis of the extract from
Moraxella sp. cells cultured in natural seawater for 10 days at
20°C, using a develosil C8 column according to the method of
Oshima et al. (1987). Mobile phase in (A) and (C) is 2 mM
1-heptanesulfonic acid in 10 mM phosphoric acid (pH 7.2).
Mobile phase in (B) and (D) is 2 mM 1-heptanesulfonic acid
in 10 mM phosphoric acid containing 10% acetonitrile (pH 7.2).
(A), GTX standards: GTX1 (5 pmoles); GTX2 (1.3 pmoles);
GTX3, (0.5 pmoles); GTX4, (12 pmoles). (B): neoSTX and
STX standards; neoSTX, (9 pmoles); STX, (5 pmoles). (C) and
(D): extract of Moraxella sp. (10 µL of extract corresponding
to cells in 5 mL of culture). Reprinted from Toxicon 28, Kodama
et al., Production of paralytic shellfish toxins by a bacterium
Moraxella sp. isolated from Protogonyaulax tamarensis. 707–
714, © 1990, Pergamon Press plc. with permission from
Elsevier.
corresponding to GTX1,4 followed by those of GTX2,3
appeared. A faint peak corresponding to neoSTX was also
observed. These results show that main toxin components
of Moraxella sp. grown under these conditions are similar to the GTXs. Figure 20 and Table 3 show the changes
of total toxin amounts per 1 L of culture and proportion
of toxin components which were calculated from the results of HPLC-fluorometric analysis. In the seawater,
Moraxella sp. produced PSP toxins within 24 h, however, the amount was low for one week, but increased
considerably by the 10th day. The pattern of toxicity
change in 1% MB was similar to that in the seawater,
although the toxicity did not increase as in the seawater.
Throughout the incubation period the toxin profile
changed (Table 3). However, the main components were
always similar to GTXs, especially GTX1,4. In the extreme cases, most of the toxin was GTX1,4. STX and
neoSTX were scarcely observed. These findings suggest
that transformation of toxin components occurs in
Moraxella sp.
doi:10.5047/absm.2010.00301.0001
17
Fig. 20. Changes of total toxin amount during culture. 䊉: culture in 1% Marine Broth; 䊊: culture in natural seawater. Reprinted from Toxicon 28, Kodama et al., Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from
Protogonyaulax tamarensis. 707–714, © 1990, Pergamon Press
plc. with permission from Elsevier.
These results show that Moraxella sp. required sodium chloride for its growth and grew best in shaken cultures, indicating that it is an aerobic marine heterotrophic
bacterium. This fact is also supported by stable growth
under various conditions in MB which is prepared from
artificial seawater. However, during 64 h, it did not produce significant amount of toxin under these culture conditions. Only cells of the standing culture in Heart Infusion (HI) at 25°C showed significant toxicity although
the culture grew poorly under this condition. Interestingly,
once produced, toxin often disappeared during incubation. Although it is poorly understood about the metabolism of PSP toxins, these findings suggest that the toxin
is an intermediate which is metabolized to non-toxic
substance(s). Chemical reactions between GTXs and biological thiols such as GSH are considered to be involved
in these phenomena. On the other hand, the toxin appeared
in MB cultures which were grown for 168 h at 25°C. The
cells at 168 h incubation should be under a starved
condition, because they were left under a nutrition deficient environment for more than 100 h. In order to obtain energy for survival under starved conditions,
Moraxella sp. possibly catabolizes its biological
substance(s). PSP toxins might be a kind of catabolites
of the metabolism (Kodama et al. 1990b).
10. Bacteria in the cells of toxic dinoflagellates
Several studies indicate that axenic, or bacteria-free,
dinoflagellate cultures retain the ability to produce these
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
18
Table 3. Toxin composition of Moraxella sp. cultured seawater and 1% marine broth.
Toxin composition*
Duration
GTX1
Culture medium
(days)
mole %
GTX2
GTX3
Natural seawater
1
2
3
4
5
6
7
10
41.0
25.6
100.0
50.5
100.0
70.5
56.7
10.0
11.8
8.0
—
8.7
—
23.7
—
0.5
2.7
2.2
—
1.8
—
5.8
—
1.4
1% Marine Broth
1
2
3
4
5
6
7
10
28.1
20.3
37.0
1.0
—
34.8
90.7
40.7
—
6.2
—
0.1
—
4.4
9.3
10.4
2.5
1.8
—
3.4
—
—
—
1.6
GTX4
neoSTX
STX
44.5
64.2
—
28.7
—
—
43.3
87.3
—
—
—
—
—
—
—
0.7
—
—
—
—
—
—
—
—
69.4
71.7
63.0
95.5
100.0
60.8
—
47.3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
*Toxin composition was calculated by comparing peak heights of samples against standard PSP toxins and expressed as
mole %.
This article was published in Toxicon, 28, Kodama, Ogata, Sakamoto, Sato, Honda and Miwatani, Production of paralytic
shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis, 707–714, © Elsevier (1990).
toxins at a level similar to those found in nonaxenic cultures (e.g. Kim et al. 1993). If bacteria are involved in
toxin production in dinoflagellates, they should be present
within the dinoflagellates. Several investigators including us have reported the direct observation of intracellular bacteria in toxic species of dinoflagellates by electron
microscopy (Silva 1979; Kodama and Ogata 1988; Franca
et al. 1996), although the bacterial numbers were generally few and such bacteria were not always discernible
even with cells of the same algal culture. Although toxigenic bacteria could be isolated from toxic dinoflagellates,
it was not clearly proven whether the isolated bacterial
strains and the corresponding intracellular bacteria were
the same.
In order to know the presence of intracellular bacteria in the toxic A. tamarense, we used an antibody
against a toxin-producing bacterium to perform Western
blots of an extract from the “host,” an A. tamarense isolate (axenic culture) (Kodama et al. 1996a). Increased
cross reactivity was observed after the apparent disruption of residual (=accumulation) bodies present within
the algal cells. Figure 21 shows the results from western
blot analysis using antibody against Moraxella sp. and
the extract of A. tamarense cells which was extracted
mildly with phosphate buffered saline (PBS) using a
Teflon homogenizer. Several faint bands were observed,
doi:10.5047/absm.2010.00301.0001
Fig. 21. Western blot analysis of the extract of Alexandrium
tamarense prepared by a Teflon homogenizer using antiMoraxella sp. antibody. A: extract of Moraxella sp., B: extract
of A. tamarense. Reprinted from Kodama et al., Symbiosis of
bacteria in Alexandrium tamarense. In: Yasumoto, Oshima and
Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996,
pp. 351–354, Fig. 1.
some of which corresponded with those of Moraxella sp.
extract, suggesting the presence of protein components
of Moraxella sp. in A. tamarense cells. However, the
number of the bands was too small to show clearly the
presence of bacterial protein components in A. tamarense
cells. As the protein components were considered to be
not sufficiently extracted, the homogenate to prepare the
extract was observed under light microscope. As shown
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 22. Microscopic observation of the homogenate prepared
by a Teflon homogenizer. Upper: light microscopic observation; lower: fluorescent microscopic observation. Reprinted
from Kodama et al., Symbiosis of bacteria in Alexandrium
tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful
and Toxic Algal Blooms. Intergovernmental Oceanographic
Commission of UNESCO, Paris. 1996, pp. 351–354, Fig. 2.
19
in Fig. 22, most of the cells were disrupted, but many
intact organelle were observed. These organelle showed
fluorescence when activated by ultraviolet light, showing that the organelle were hard to be homogenized
by the method applied. Therefore, homogenate of
A. tamarense was sonicated for 1, 5 and 10 min in phosphate buffered saline (PBS) in the same buffer containing 2% SDS. Figure 23 shows the observation of the
homogenates under fluorescent microscope. Although
most of the cells were disrupted by 1 min sonication, many
fluorescent organelle were not disrupted by the procedure in both buffers. Significant number of these organelle were still observed even after 10 min sonication. However, the number decreased considerably, showing that they were disrupted as the sonication time became longer (Fig. 23). SDS was effective for the disruption of the organelle. Western blot analysis of the extracts
prepared from these homogenates showed that the staining of the bands with antibody against Moraxella sp. became stronger as the sonication time of the cells in SDScontaining buffer for longer time was effective to extract
the fluorescent organelle. Therefore, a large number of
the cells (5 × 107 cells) were extracted with 0.5 mL of
1.25 M tris containing 10% SDS to prepare the concentrated extract. The extract thus obtained showed clear
bands corresponding to almost all the protein components
of Moraxella sp. in western blot analysis (Fig. 24). These
results show that protein components which reacted with
antibody against protein extracts of Moraxella sp. are
contained in these fluorescent organelle. A. tamarense
Fig. 23. Fluorescent microscopic observation of
homogenates prepared under different conditions.
Upper: Cells were sonicated for 1, 5, 10 min in
SDS-containing buffer; lower: Cells were sonicated
for 1, 5, 10 min in PBS. Bar: 100 µm. Reprinted
from Kodama et al., Symbiosis of bacteria in
Alexandrium tamarense. In: Yasumoto, Oshima and
Fukuyo (eds). Harmful and Toxic Algal Blooms.
Intergovernmental Oceanographic Commission of
UNESCO, Paris. 1996, pp. 351–354, Fig. 3.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
20
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 24. Western blot analysis of the extract of Alexandrium
tamarense prepared by sonication in SDS-containing buffer.
A: extract of Moraxella sp.; B: extract of A. tamarense. Reprinted from Kodama et al., Symbiosis of bacteria in
Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo
(eds). Harmful and Toxic Algal Blooms. Intergovernmental
Oceanographic Commission of UNESCO, Paris. 1996, pp. 351–
354, Fig. 4.
clone used in the present study was grown and maintained for more than a year under axenic condition. No
bacteria were observed in the cell-free medium of the
clone in monthly test for detection of bacteria. Therefore, the presence of bacterial substances in the extract
of A. tamarense clone implies that bacteria are living in
the cells, probably in the fluorescent organelle.
Figure 25 shows the light and fluorescent
microscopy of A. tamarense cell harvested at late stationary phase. Autofluorescent organelle is seen under the
nucleus. The organelle showed blue-white fluorescence
under UV light excitation, green fluorescence under blue
light excitation with a red cut-off filter and yellow without the red cut-off. These characteristics are similar to
those of autofluorescent organelle called PAS/accumulation bodies observed in other dinoflagellates which are
considered to be lysosome. Franca et al. (1995) and
Mascarenhas et al. (1995) have suggested that residual
bodies of A. lusitanicum which seem to be the same as
PAS/accumulation bodies, contain remnant of bacteria.
They observed living bacteria in the space between the
outer cellular membrane and the thecal complex, which
seem to originate from environment. From these facts,
they suggested that incorporated bacteria are trapped by
food vacuole and transferred to the residual body to be
digested. The digested bacteria might be a nutritive source
for Alexandrium. On the contrary, the results of the present
study show that bacteria are living in the Alexandrium
residual body. Therefore, a part of the bacteria incorporated into the inside the cells seem to survive and grow in
the organelle while others are digested in the organelle.
It is well known that organic substances such as soil extracts and vitamins are essential for the growth of
Alexandrium. We suggest that A. tamarense can grow by
utilizing bacteria as well as organic substances under limitation of nutrient or light intensity (Ogata et al. 1996).
These findings suggest that Alexandrium spp. require organic substances which originate from bacteria. Under
doi:10.5047/absm.2010.00301.0001
Fig. 25. Microscopic observation of Alexandrium tamarense
cell. Left: light microscopic observation; right: fluorescent
microscopic observation. Reprinted from Kodama et al., Symbiosis of bacteria in Alexandrium tamarense. In: Yasumoto,
Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms.
Intergovernmental Oceanographic Commission of UNESCO,
Paris. 1996, pp. 351–354, Fig. 5.
axenic condition in which no bacteria are supplied from
the environment, a part of bacteria growing in the lysosome-like organelle are considered to be digested in the
organelle to provide an organic substance to A. tamarense
for its growth. The relation between A. tamarense and
endocellular bacteria may be mutualism.
These results and the facts that bacteria could be
isolated from an axenic culture of this dinoflagellate only
after disruption of the residual bodies, led us to contend
that intracellular bacteria exist within the residual bodies
and these retain the ability to reproduce within the algal
cells. This idea is consistent with electron microscopic
observations showing partially digested bacteria in the
residual bodies of several toxic dinoflagellates (Franca
et al. 1995). The fact that apparently stable association of
certain bacteria with an algal isolate is observed over many
years also support the idea. Generally, bacteria responsible for infection are killed by defense mechanisms of the
host organism. However, some specific infectious bacteria are known to possess escape mechanisms allowing them to survive within cells of the host (Groisman
1992). It is hypothesized that the growth of such bacteria
inside residual bodies may be controlled by digestive processes, thereby harmonizing bacterial growth with that of
the dinoflagellate and allowing extended maintenance of
the intracellular-bacterial flora.
11. Utilization of organic substances for growth and
toxin production by Alexandrium tamarense
There have been many dinoflagellate species reported to possess a heterotrophic behavior such as
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 26. Growth curve of Alexandrium tamarense cultured
semicontinuously in N-limited T1 medium in which nitrate level
was reduced to 2% of the normal medium. A half volume of
culture was diluted with equal volume of fresh medium successively when the cell density increased to double. Reprinted
from Ogata et al., Utilization of organic substances for growth
and toxin production by Alexandrium tamarense. In: Yasumoto,
Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms.
Intergovernmental Oceanographic Commission of UNESCO,
Paris. 1996, pp. 343–346, Fig. 1.
auxotrophy or phagotrophy (Gaines and Elbrachter
1987). However, dinoflagellates which produce PSP
toxins such as A. tamarense have until now been regarded
as photosynthetic organisms. Therefore, their growth
physiology and toxin production have been studied
from the standpoint that they are autotrophic organisms.
As described above, we gave a clear evidence that
photosynthesis is necessary for the toxin production of
A. tamarense (Ogata et al. 1987b). The fact also supports
the autotrophic character of A. tamarense. On the other
hand, Alexandrium spp. require some organic substances
such as vitamins (Iwasaki 1979), indicating that they also
possess heterotrophic characters. However, little is
known about this process on these species. We found that
A. tamarense possesses bacteria in the cells and that the
isolated bacteria produce toxins, though the productivity
is low (Kodama et al. 1988). Based on these results, we
propose a hypothesis that these bacteria are necessary for
A. tamarense to produce toxins. Thus it is based on a
premise that A. tamarense utilizes organic substance. Such
heterotrophic character is considered to be also important to understand the growth of these species in the natural environment. Despite numerous reports on the relationship between the occurrence of Alexandrium spp. and
environmental factors such as inorganic nutrient concentrations, it seems difficult to correlate the growth of this
dinoflagellate solely to these factors. The utilization of
organic substance(s) of A. tamarense was studied in relation with growth and toxin production of this species
(Ogata et al. 1996).
The unialgal and axenic cultures were maintained under irradiation of 60 µmol photons·m –2·s–1
(cool-white fluorescent lamps) with 16 h:8 h LD cycle.
doi:10.5047/absm.2010.00301.0001
21
T1 and SWII medium (nitrate; 0.71 mM) (Iwasaki 1961)
were used for unialgal and axenic cultures, respectively.
Semi-continuous culture was established by repeating
dilution of culture with an equal volume of fresh medium
when the cell density increased to the double. An aliquot
of the cells was taken to measure cell density under a
microscope. The growth rate (µ 2) was estimated by the
method of Fukazawa et al. (1980). Toxin contents of
the cultured cells were analyzed by HPLC according
to Oshima (1995b). As shown in Fig. 26, the semi-continuous culture acclimated to N-limited condition was established by cultivating A. tamarense in N-limited T1
medium in which nitrate was reduced to 2% of the normal medium. During the first 30 days, the growth of
A. tamarense was unstable, but it showed stable growth
with growth rate of 0.25 division/day after 30 days. The
culture maintained exponential phase, showing that cells
grew continuously under N-limited condition. As shown
in Fig. 27, toxin content of the cell dropped to about 1/10
that of those in the initial culture cultivated in normal T1
medium as indicated previously (Boyer et al. 1987;
Anderson et al. 1980). When nitrate (1.0 mM) or yeast
extract (DIFCO; 20 mg·L–1) was added to the culture (indicated by arrows in Fig. 27), the cells grew well. The
maximum yield of the culture increased remarkably in
both cases. At the same time, toxin production of the cells
was enhanced to almost 5 times by these treatments. Nitrate and ammonia in yeast extract used here were measured to be 0.055 and 1.095 µmol·g–1, respectively, being too low to explain the growth after the addition of
yeast extract. These results show that A. tamarense can
use organic N-substance in yeast extract as the nitrogen
source instead of nitrate.
Figure 28 also shows the effect of bacterium,
Moraxella sp., on the growth and toxin production of
A. tamarense cultured under N-limited condition semicontinuously. At the dilution of the culture in the maximum density, 107 cells of live or autoclaved Moraxella
sp. cells were added with 100 mL of the fresh medium.
The maximum cell number of A. tamarense increased
significantly by addition of autoclaved bacterial cells,
as in the case of yeast extract. This increase in growth
was repeatedly observed. Toxin production of cells
was also induced by this treatment. In contrast, growth
of A. tamarense became lower by the addition of live
Moraxella sp. cells. However, when the addition of bacterial cells was repeated, A. tamarense started to grow
well as in the case of autocraved cells (0.39 division/day).
Toxicity of cells also increased gradually by the treatment. These results indicate that A. tamarense also possess phagotrophic characters and utilize environmental
bacteria for their growth and toxin production. As mentioned above, Franca et al. (1995) observed bacteria in
the space between the outer cellular membrane and the
thecal complex, and suggested that these bacteria might
© 2010 TERRAPUB, Tokyo. All rights reserved.
22
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 27. Change of growth (lower) and toxin production (upper) of Alexandrium tamarense after addition of nitrate (1.0 mM)
or yeast extract (20 mg·L–1) under N-limited condition. Nitrate and yeast extract were added at a time indicated by arrow.
N: Culture supplemented with nitrate, Y: Culture supplemented with yeast extract, B: Control culture. Reprinted from
Ogata et al., Utilization of organic substances for growth and toxin production by Alexandrium tamarense. In: Yasumoto,
Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO,
Paris. 1996, pp. 343–346, Fig. 2.
Fig. 28. Change of growth (lower) and toxin production (upper) of Alexandrium tamarense after addition of autoclaved
(AM) or live (LM) Moraxella sp. cells under N-limited condition. Bacterial cells were added at times indicated by arrows.
Reprinted from Ogata et al., Utilization of organic substances
for growth and toxin production by Alexandrium tamarense.
In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic
Algal Blooms. Intergovernmental Oceanographic Commission
of UNESCO, Paris. 1996, pp. 343–346, Fig. 3.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
23
be a nutritive source of the dinoflagellate. Our results
support their observations.
Previously, we have reported that de novo synthesis of toxins in A. tamarense is repressed under low
light condition (Ogata et al. 1987b). Therefore, the effect of organic substance on the toxin production under low light condition was examined. As shown in
Fig. 29, cell division of A. tamarense stopped at midexponential phase by lowering light intensity from 60
to 5 µmol photons·m–2·s–1. No significant toxin production was observed during 4 days under this condition.
When the yeast extract (20 mg·L–1) was added to the culture at the time of lowering light intensity, cells were observed to keep growing though the growth rate became
lower. During the period, the toxin contents of cell increased. These results suggest that A. tamarense can
grow by utilizing organic substances such as yeast extract, if they are available, even when it can not grow
autotrophically under low light condition.
Cultures used in these experiments were not axenic.
It could be possible that bacteria contaminating the culture decomposed the added organic substances to inorganic nutrients which are available for A. tamarense.
Therefore, the effect of organic substance on the growth
was examined by using an axenic culture. As shown in
Fig. 30, the axenic strain grew well in SWII medium with
the maximum yield of 8 × 104 cells·mL–1. Cells grown
in N-depleted SWII medium (SWII-N) showed a similar growth, but the maximum yield was much depressed
to 2 × 10 3 cells·mL–1. When the culture was spiked with
nitrate (1.0 mM) at the stationary phase, N-limited culture induced remarkable cell division, resulting in the recovery of cell yield of 8 × 104 cells·mL–1. Growth of the
cells in SWII-N was also induced by the addition of
yeast extract (20 mg·L–1), though the growth rate and cell
yield were lower than those of nitrate addition. These results clearly show that A. tamarense is mixotrophic organism. It can grow autotrophically under the conditions
suitable for photosynthesis. When the environment is not
suitable for photosynthesis, it can grow using energy
Fig. 29. Changes of growth (lower) and toxin production (upper) of Alexandrium tamarense by addition of yeast extract
(20 mg·L–1) under light limited condition. Culture was divided
to two parts after lowering light intensity at mid-exponential
phase. A: Control culture, B: culture supplemented with yeast
extract at the time indicated by arrow. Reprinted from Ogata et
al., Utilization of organic substances for growth and toxin production by Alexandrium tamarense. In: Yasumoto, Oshima and
Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996,
pp. 343–346, Fig. 4.
Fig. 30. Effect of supplemented yeast extract on the growth of
axenic Alexandrium tamarense cells. Cells were grown in SWII
medium (䊐), N-depleted SWII (䉱), SWII supplemented with
nitrate (1.0 mM) (䉬), and SWII supplemented with yeast extract (20 mg·L–1) (䊊). Nitrate and yeast extract were added at
the time indicated by arrow. Reprinted from Ogata et al., Utilization of organic substances for growth and toxin production
by Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo
(eds). Harmful and Toxic Algal Blooms. Intergovernmental
Oceanographic Commission of UNESCO, Paris. 1996, pp. 343–
346, Fig. 5.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
24
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
heterotrophically supplied. It is striking that A. tamarense
produces toxins even when it grows heterotrophically.
This fact is very important to understand the blooming
mechanism of Alexandrium species.
Although a number of survey have been conducted
on the environmental factors for the growth of natural
population of A. tamarense, little is known about their
blooming mechanisms. In the field survey in Ofunato Bay,
the bloom of A. tamarense was often observed under the
environment in which the level of inorganic N such as
nitrate or ammonia was low (unpublished data). During
the bloom, this species was found in higher densities at
around 10 m depth where light intensity was as low as
30 µmol photons·m–2·s–1 even during sunny day. These
facts indicate that natural population of A. tamarense
grows in an environment in which its autotrophic activity is limited. Jacobsen and Anderson (1996) presented
electron micrographs clearly showing that A. ostenfeldii
ingests the plankton as food. These findings suggest that
heterotrophic characters as well as autotrophic ones play
an important role for the growth and toxin production of
the natural population of Alexandrium species.
12. Toxin-producing bacteria in marine ecosystem
Since the first finding that a bacterium (PTB-1) isolated from A. tamarense produces STX (Kodama et al.
1988; Doucette and Trick 1995), several strains of STXproducing bacteria have been isolated from dinoflagellates
(Franca et al. 1996; Gallacher et al. 1997). Also we have
detected STXs in fractions of particles smaller in size than
dinoflagellates (Sakamoto et al. 1993).
In a monitoring survey on shellfish toxicity, we
analyzed STXs in particles suspended in seawater after
fractionation of the particles according to size by passing
them through sieves of different pore sizes. Figure 31
shows data obtained for samples from Tanabe Bay where
shellfish became toxic during a bloom of A. catenella
(Sakamoto et al. 1992, 1993). Surprisingly, no significant amount of shellfish toxicity increased considerably
(indicated by arrows in Fig. 31). In contrast, significant
toxicity was detected in association with particles smaller
than A. catenella such as those in the size ranges of 5–20
and 0.45–5 µm by HPLC analysis. The 5–20 µm fraction
showed the highest level of toxicity when the cell density of A. catenella was very low. The toxins in this
fraction were highly purified and identified as STX by
mass spectrometry. These findings clearly indicate that
an unknown STX-producing organism(s), smaller than
A. catenella, occurs together with A. catenella in the
seawater. Similar results were obtained for samples from
Ofunato Bay during a bloom of A. tamarense (Kodama
et al. 1990a). A slight increase in shellfish toxicity was
observed when fractions containing particles smaller than
dinoflagellates showed considerable toxicity. As no significant toxicity was observed in other particle fractions,
doi:10.5047/absm.2010.00301.0001
the shellfish toxicity during the period seemed to be due
to the toxins associated with these particles. However,
shellfish toxicity did not increase remarkably, although
high levels of STXs were detected in the fractions containing particles with a size smaller than dinoflagellates.
It seems quite strange that shellfish toxicity increased significantly as A. catenella grew to high density; yet no
significant amount of toxin was detected in A. catenella
(Sakamoto et al. 1993).
Although definitive proof of a bacterial source of
the toxin is lacking, STXs detected in particles smaller in
size than dinoflagellates are thought to originate from
bacteria grown in seawater, and thus the particles may be
living on the surface of detritus which is favorable for
their growth (Kodama et al. 1990a). Probably, as the large
number of bacterial cells are attaching on the detritus,
the amount of toxin in the detritus is often greater than
the amount associated with free-living bacteria. However,
even though the detritus fraction (5–20 µm fraction)
showed high toxicity, shellfish toxicity did not increase
remarkable. These findings may indicate that bacterial
toxins are not directly associated with shellfish toxicity.
It is also noteworthy that, although shellfish became toxic
during a bloom of A. catenella, no significant toxicity
was detected in the A. catenella cells from this location
(See Fig. 31). These findings support the idea that the
toxicity of shellfish cannot be attributed solely to transport of dinoflagellates toxins via the food chain. Toxinproducing bacteria may possess “biological component”
in which toxin is incorporated as a part; the “biological
component” which releases toxin by digestion.
The distribution of STX-producing bacteria in the
marine environment appears to be quite widespread. Investigators in increasing numbers are now reporting the
isolation of such bacteria from locations that are spatially
and temporally distinct from the locations of the
dinoflagellates. Levasseur et al. (1996) examined the
potential for bacterial production of STXs in the St. Lawrence estuary, Canada. They screened bacterial strains
isolated from the St. Lawrence estuary by HPLC and
found four strains positive for STXs, indicating that putatively toxigenic bacteria are present in that region.
Interestingly, these four bacterial strains isolated from
the St. Lawrence estuary by Levasseur et al. (1996) originated from sites exhibiting no concurrent PSP activity,
and three of these locations had no prior history of bloom
formation. Although free-living and particle associated
bacteria capable of producing STXs appear to exist in
the St. Lawrence estuary, only the toxins associated with
larger size fractions accounted for shellfish toxicity during this Alexandrium bloom. These results indicate that
STX-producing bacteria ingested by shellfish could not
be directly responsible for the STXs in the shellfish, although a contribution of toxin-producing bacteria to
shellfish toxicity could not be ruled out.
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 31. Seasonal variation of Alexandrium catenella abundance, scallop toxicity and the toxicity of particle fractions from
January to June 1990 in Tanabe Bay. (A) Vertical distribution of A. catenella. Cell density (cells L–1): (䊐) 0 to 99; (䊐) 100 to
999; (䊐) 1000 to 4999; (䊐) 5000 to 9999; (䊐) >10000. (B) Abundance of A. catenella (䊉) (no of cells) and scallop toxicity
(䊊, MU g–1). A. catenella were collected from 1 L samples taken every 2 m from the surface to 8 m depth. Arrows: peaks of
A. catenella abundance where shellfish toxicity is decreasing. (C) Toxicity of particle fractions: (䊐) > 20 µm; (䉱) 5–20 µm;
() 0.45–5 µm. Reprinted with permission from Marine Ecology Progress Series, 89, Sakamoto et al., Causative organism of
paralytic shellfish toxins other than toxic dinoflagellates, 229–235, Fig. 1, © 1992, Inter-Research.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
25
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M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Information on the distribution of STX-producing
bacteria in marine ecosystems is important to solve the
problem of the association between these bacteria and
the STXs of shellfish and other organisms. In order to
prove the toxigenicity of bacteria, more detailed chemical evidence such as analytical data obtained by mass
spectrometry or nuclear magnetic resonance (NMR) are
required. However, because of the low toxin productivity of bacteria, various investigators have applied indirect methods for toxin identification such as HPLC and
bioassay of crude extracts. When crude extracts are
analyzed by fluorometric-HPLC, ghost peaks with retention times identical to those of standard toxins often appear (Onodera et al. 1997). However, these peaks can be
distinguished from toxin peaks by changing the reaction
reagent for post-column derivatization to water; the ghost
peaks do not disappear as a result of this treatment, but
the toxin peaks disappear. In addition, these impurities
can be mostly removed by pretreatment of the samples,
such as by partial purification of the toxins by Bio-Gel P2 (Bio-Rad Laboratories, Richmond, CA) or activated
charcoal treatment. HPLC analysis of bacterial toxins after
such treatment will provide more detailed information
concerning the toxigenicity of the bacteria.
13. STXs in organisms other than dinoflagellates and
their associated organisms
It is well known that STXs are found not only in
dinoflagellates and their associated shellfish but also in
phylogenically diverse species of animals. Noguchi et al.
(1969) showed for the first time that the xanthid crab
Atergatis floridus, a non-plankton feeder animal accumulates a high level of STX which could not be associated
with dinoflagellates. After this discovery, high levels of
STXs were detected in various animals, such as the horseshoe crab, Carcinoscorpius rotundicauda (Fusetani et al.
1982) and several species of marine snails (Kotaki et al.
1981). We also found that a low level of STX is detected
in puffer Takifugu pardalis in addition to a large amount
of tetrodotoxin (TTX) (Kodama et al. 1983a). In 1988, a
food poisoning including fatal cases by ingestion of freshwater puffer Tetraodon fangi occurred (Laobhripart et al.
1990). We analyzed the puffer by mouse assay and found
that the puffer possesses a potent neurotoxin, though no
TTX was detected by chemical analysis (Sato et al. 1997).
In the analysis of PSP toxins by HPLC, however, a large
amount of a toxin was detected instead of TTX which
was identified as STX (Sato et al. 1997) (Fig. 32). Although no intensive survey on the PSP toxins-bearing
organisms was conducted, toxins were not detected in
freshwater bivalves collected from the area where the
puffer were observed. Thus, the source of STX found in
freshwater puffer is unknown.
In order to know the occurrence of STXs in puffer,
we surveyed toxins in tropical species of puffer on which
there is few knowledge (Sato et al. 2000a). Seven species of puffer including 27 specimens of Arothron
Fig. 32. Fast bombardment mass spectrum of freshwater puffer toxin. A fast atom bombardment mass spectrum (FAB-MS) of
the purified toxin was measured in glycerol as a matrix through a magnetic scan from 25 to 500 mass units. The toxin
exhibited an ion peak at m/z = 300, 282 and 374, which correspond with a pseudomolecular ion (M + H)+ of authentic STX,
(M + H – H2O)+ and (M + H + glycerol – H2O)+, respectively. Reprinted from Toxicon, 35, Sato, Kodama, Ogata, Saitanu,
Furuya, Hirayama and Kakinuma, Saxitoxin as a toxic principle of a freshwater puffer Tetraodon fangi, in Thailand, 137–140
© 1997, Pergamon Press plc. with permission from Elsevier.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
27
Fig. 33. Toxicity of various organs of puffers collected from Masinloc Bay, Philippines. The toxicity was measured by mouse
bioassay and expressed as mouse unit (MU) of TTX. L: liver, I: intestine, M: mussel, S: skin. (a) Number of specimens which
did not kill mice (ND): 2, (b) 2, (c) 1, (d) 3, (e) 1, (f) 2, (g) 1, (h) 5, (i) 1. Reprinted from Toxicon, 38, Sato, Ogata, Borja,
Gonzales, Fukuyo and Kodama. Frequent occurrence of paralytic shellfish poisoning toxins as dominant toxins in marine
puffer from tropical water, 1101–1109, © 2000, Pergamon Press plc, with permission from Elsevier.
mappa, 25 of A. manillensis, 20 of Chelonodon patoca,
6 of A. nigropunctatus, 5 of A. hispidus, one A. stellatus
and one A. reticularis were collected at Masinloc Bay,
Luzon, Philippines over the year of 1992. The tissues
excised from partially thawed specimens were subjected
to extraction and analyzed by HPLC for PSP toxins as
well as TTX. Figure 33 shows the toxicity of the extracts
analyzed by mouse bioassay. Most of the extracts killed
mice with typical signs of TTX and STXs. In the chemical analysis, a considerable level of STXs was detected
in addition to TTX. Although the number of the specimens was not enough, the major toxin of these puffer
seemed to be STXs. In Fig. 34 is shown one of the results
obtained from A. mappa specimen by HPLC analysis. In
addition to TTX, significant level of neoSTX,
decarbamoyl STX, GTX5 and STX were detected. Most
of the puffer specimens showed similar profiles, suggesting that the major toxin in puffer Masinloc Bay is
STX (Fig. 35).
The Masinloc Bay where these puffer specimens
were collected is well known for the bloom of Pyrodinium
bahamense var. compressum, a tropical PSP toxinsdoi:10.5047/absm.2010.00301.0001
Fig. 34. HPLC-chromatograms of a skin extract of A. mappa.
(A) Analysis by HPLC specially designed for TTX. For detail conditions see Yotsu et al. (1989). (B) and (C) analysis by
HPLC specially designed for PSP toxins. For detail conditions
see Oshima (1995a). Reprinted from Toxicon, 38, Sato, Ogata,
Borja, Gonzales, Fukuyo and Kodama. Frequent occurrence of
paralytic shellfish poisoning toxins as dominant toxins in marine puffer from tropical water, 1101–1109, © 2000, Pergamon
Press plc, with permission from Elsevier.
© 2010 TERRAPUB, Tokyo. All rights reserved.
28
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 35. Mol% of PSP toxins to total toxin (TTX + PSP toxins) in each organ of puffer. L: liver, I: intestine, M: muscle,
S: skin. Reprinted from Toxicon, 38, Sato, Ogata, Borja,
Gonzales, Fukuyo and Kodama. Frequent occurrence of paralytic shellfish poisoning toxins as dominant toxins in marine
puffer from tropical water, 1101–1109, © 2000, Pergamon Press
plc, with permission from Elsevier.
producing dinoflagellate (Montojo et al. 2006). Bivalve
in the bay become toxic almost every year. Interestingly, the toxin profile of puffer was close to that of
P. bahamense var. compressum and its associated
bivalves. On the other hand, Zaman et al. (1997) reported
that toxin profile of freshwater and/or brackishwater
puffer Tetraodon cutctia and C. patoca collected from
the rivers and tributary in Dhaka, Bangladesh, was also
close to that of P. bahamense var. compressum, though
P. bahamense var. compressum can not grow in freshwater. Thus the PSP toxins found in puffers from
Masinloc Bay was not derived from P. bahamense
var. compressum. The source of STXs found in the Philippine puffers is unknown.
Yasumoto et al. (1981) concluded that the STXs in
the xanthid crab are not derived from dinoflagellates, as
no STXs were detected in bivalves living in the same
area where the toxic xanthid crab is inhabiting. Instead,
they detected a low level of STXs in Jania sp., a
carcacious alga which is often found in the stomach of
the toxic xanthid crab, and suggested that the origin of
STXs in the xanthid crab is this alga (Kotaki et al. 1983).
It is possible that the xanthid crab accumulates a high
level of STXs by continuous ingestion of Jania sp. over
doi:10.5047/absm.2010.00301.0001
a long period. Meanwhile, the xanthid crab is known to
release STXs (Noguchi et al. 1985). Arakawa et al. (1998)
showed that a large proportion of the STXs injected into
the xanthid crab disappeared within a short period. This
finding implies that the rate of release of STXs from the
xanthid crab is significantly high. Therefore, it is unlikely
that the xanthid crab accumulates a high level of STXs
by continuous ingestion of Jania sp. with a low concentration of STX, even over a long period.
Interestingly, these STX-bearing animals often
possess tetrodotoxin (TTX) as well, a potent neurotoxin
which binds to the same biological receptor as STXs
(Kao 1966). This suggests that organisms known to
possess either STXs or TTX may possess both of these
toxins. This speculation is supported by the fact that a
significant amount of TTX has been detected in A.
tamarense, a representative dinoflagellate capable of producing STXs. During a bloom of A. tamarense, considerable amounts of TTX accumulate in shellfish together
with STXs (Kodama et al. 1993). As shown in Fig. 35
the levels of TTX in the shellfish was shown to be parallel with the levels of STXs (Kodama et al. 1993). These
findings indicate that TTX found in the shellfish originates from A. tamarense. Actually, a significant level of
TTX was identified in cultured cells of A. tamarense
(Kodama et al. 1996b). This is the first case to be shown
that A. tamarense could be the causative organism for
TTX-bearing animals.
It is well known that TTX is produced by bacteria,
although the productivity is very low (Noguchi et al. 1986;
Yasumoto et al. 1986). It is noteworthy that TTXproducing bacteria and STX-producing bacteria display
similar characteristics in terms of toxin productivity. The
toxin productivity of TTX-producing bacteria is very low
and the TTX levels in the TTX-producing bacteria increase significantly under starvation conditions (Noguchi
et al. 1986; Yasumoto et al. 1986). Although the evidence
is circumstantial, various phenomena suggest that these
bacteria are widely distributed in natural ecosystems. The
origin of the STXs in toxic animals not associated with
dinoflagellates seems to be bacteria, as in the case of TTX,
and the mechanism by which the organisms become toxic
owing to the presence of TTX and STXs derived from
bacteria seems to be similar in each instance.
14. Possible mechanism by which pufferfish become
toxic: a model based on the mechanism by which
accumulation of STXs occurs in dinoflagellates
It is well known that wild puffer possess a potent
neurotoxin named as tetrodotoxin (TTX). Generally, TTX
is contained in various toxic species of puffer. However,
TTX is not always contained in all the specimens of toxic
species. Non-toxic specimens are often observed among
the toxic species of puffer. It has been a mystery why
puffer possess the potent toxin, TTX. Although the
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
reason is still not clear, it is known by fish culturists that
puffer which are artificially hatched and fed an artificial
diet are nontoxic. Matsui et al. (1982) demonstrated that
nontoxic cultured puffer Takifugu rubripes become toxic
when fed a diet containing TTX, and indicated that TTX
in the puffer is an exogenous substance which originates
from the diet. On the other hand, TTX has been shown to
be produced by bacteria (Noguchi et al. 1986; Yasumoto
et al. 1986) and thus the source of TTX in TTX-bearing
animals such as puffer is considered to be bacteria. Hence,
TTX of bacterial origin is considered to accumulate in
the puffer via food webs (Yasumoto et al. 1992). The
screening of various marine organisms for TTX revealed
that this toxin is widely distributed in marine organisms,
some species of which could be components of the diet
of puffer (Noguchi et al. 1983; Miyazawa et al. 1986).
These studies also showed that the trumpet shell Charonia
sauliae accumulates TTX by ingesting the TTX bearing
starfish Astropecten polyacanthus (Narita et al. 1984).
However, such a case clearly supporting the food chain
theory of TTX accumulation in toxic animals is rare. The
distribution of toxic components of the diet of the puffer
rarely overlaps with that of toxic puffer, indicating that
the puffer toxin cannot be accumulated via a simple food
web mechanism. On the contrary, Noguchi et al. (1987)
proposed that puffer become toxic by absorbing a low
level of TTX produced by intestinal bacteria over a long
period. This idea seems plausible, because TTXproducing Vibrio alginolyticus is a dominant species in
the intestinal bacterial flora of toxic puffer (Noguchi et
al. 1987). However, Sugita et al. (1988) reported that
V. alginolyticus is also a dominant member of the intesti-
nal bacterial flora of nontoxic cultured puffer T. rubripes.
We analyzed TTX levels in the liver, the main toxic organ of the puffer, and in the intestine of cultured puffer
specimens of 60 days and 6 months old, applying a sensitive method of analysis of TTX (Sato et al. 1990). No
TTX was detected in the liver of either juvenile or adult
puffer specimens, whereas a low level of TTX, 0.2 mouse
unit (MU)·g–1 (where one MU is a dose required to kill a
20-g male mouse within 30 min), apparently due to intestinal bacteria, was detected in the intestine of both samples. These observations show that the level of TTX in
the intestine is too low for accumulation by the puffer.
TTX- and STX-bearing animals have been reported to be resistant to these toxins (Koyama et al.
1983). However, these animals die when a substantial
amount of the toxin (about 1000 times the lethal dose for
common animals) is injected (Koyama et al. 1983). These
observations show that the toxins are also toxic to these
animals. Although the mechanism is not known, puffer
sometimes accumulate TTX at levels much higher than
their lethal dose (Kodama et al. 1984). In this regard,
species of puffer are secreting this dangerous toxin into
environmental water continuously (Kodama et al. 1985).
As shown in Fig. 36, puffer possess developed TTX-secreting glands in the skin which consist of specific TTXsecreting cells (TTX cells) (Kodama et al. 1986). When
stimulated, the puffer secretes a considerably high amount
of TTX into the environment, that is, 4 mg of TTX is
often secreted on a single stimulation (Kodama et al.
1985), showing that the rate of TTX secretion is significantly high. A similar phenomenon is also observed in
the case of the TTX bearing newt (Shimizu and Kobayashi
Fig. 36. Light micrographs of the skin of T. pardalis. (A) Longitudial section of a large alveolar secretory gland, with an
opening (arrow) to the skin surface. The cytoplasm of the scretory cells (TTX cells) are almost empty. The bar equals 100 µm.
(B) Epidermis, with mucous cells, sacciform cells and free TTX cells. The bar equals 10 µm. Helly’s fluid, PAS—hematoxylin
stain. SG, secretory gland; E, epidermis; D, dermis; L, lumen of the gland; MC, mucous cell; FC, free TTX cell. Reprinted
from Toxicon, 24, Kodama, Sato, Ogata, Suzuki, Kaneko and Aida, Tetrodotoxin secreting glands in the skin of puffer fishes.
819–829, © 1986, Pergamon Journal Ltd. with permission from Elsevier.
doi:10.5047/absm.2010.00301.0001
29
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M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
1983). These findings indicate that TTX-bearing animals
actively release TTX from a specifically developed organ and the rate of release of the toxin is significantly
high. Given that the TTX present in the puffer is accumulated via food webs, the rate of uptake of TTX should
be greater than the high rate of release, that is, a considerably large amount of TTX must be supplied to the
puffer continuously. This seems consistent with our results indicating that the cultured puffer T. rubripes did
not accumulate TTX, although a low level was detected
in the intestine. The supply of TTX from the intestinal
bacteria was too low for accumulation by the puffer.
Watabe et al. (1987) reported that tritium-labeled
TTX injected intraperitoneally into toxic puffer is accumulated in the liver for a short period and then transported to the skin and gallbladder to be released. About
70% of the injected TTX disappeared within 6 days. We
also observed that TTX injected into the nontoxic cultured puffer T. rubripes, was accumulated in the liver first,
moved to the skin, and then was released into the environment (unpublished data). About 50% of the injected
TTX disappeared within a few weeks. These data show
that TTX taken up by the puffer is not retained for a long
period because of the high rate of secretion of TTX. Although no significant amount of TTX has been detected
in the diet of the puffer, some specimens of wild puffer
accumulate substantial amounts of TTX (Kodama et al.
1984). These observations lead us to contend that puffer
accumulate TTX not via food webs but through some
other mechanism which remains unknown.
Interestingly, a considerable amount of TTX was
detected in the muscle of cultured puffer T. rubripes specimens 2 weeks after injection of TTX (unpublished data).
Watabe et al. (1987) also observed that injected tritium
labeled TTX is detected in significant concentrations
in the muscle of the puffer. In contrast, no TTX is detected in the muscle of wild specimens of T. rubripes.
Tani (1945) reported that the muscle of T. rubripes is
nontoxic, although considerable toxicity is often detected
in the liver and other organs. Based on analyses of the
toxicity of various specimens, the muscle of this species
has been designated as a safe food after careful removal
of the organs prone to contamination with the toxin by
licensed chefs in Japan. The results of these toxicity analyses indicate that natural specimens of T. rubripes never
take in such an amount of TTX at once as that administered in the experiments. The results also tend to support
the view that TTX in the puffer is not accumulated via
food webs.
As described above, we have suggested that symbiotic bacteria in dinoflagellates are involved in the production of substantial amounts of STX. Most of the toxic
species of puffer possess STXs as well as TTX (Sato et
al. 1993). In specimens of the freshwater puffer, Tetraodon
fangi, which caused food poisoning including lethal cases
in Thailand, only STX was detected as the toxic agent
doi:10.5047/absm.2010.00301.0001
(Sato et al. 1997). Therefore, the approach taken in investigation of toxins from dinoflagellates was applied to
puffer toxin, that is, attempts were made to isolate bacteria from the liver tissue of toxic and nontoxic specimens
of puffer. Interestingly, bacteria were isolated only from
toxic specimens (Kodama et al. 1995). Polyclonal antibody against the bacterium isolated from the liver reacted
with an extract of the liver from which the bacterium was
isolated. Electron microscopic observations also supported the view that bacteria were present in the liver
cells (Fig. 37). An extract of the isolated bacteria was
found to display sodium channel blocking activity and
gave a TTX-like peak on HPLC analysis. These findings
suggest that bacterial symbiosis in the liver cells is involved in the mechanism of production of puffer toxin as
in the case of toxic dinoflagellates. Most of the bacteria
isolated from the liver of the puffer were gram-positive,
which are usually not found in seawater but rather in the
sediments (Kodama et al. 1995). Polyclonal antibody
against the bacterium isolated from the liver reacted with
an extract of the liver from which the bacterium was isolated (Fig. 38). It is well known that puffer tend to hide
themselves in the bottom sediment (Katayama and Fujita
1965). It seems plausible that bacterial infection may
occur in this habitat of the puffer. This may explain why
puffer cultured in a cage, having no contact with bottom
sediments, are nontoxic.
As described above, bacteria which produce STXs
or STX-like substances are widely distributed in natural
ecosystems. This is also true for bacteria which produce
TTX or TTX-related compounds (Noguchi et al. 1987;
Simidu et al. 1990). Shewanella alga, the species found
to produce TTX, was shown to produce STXs as well
(Doucette et al. 1988). This species shows wide
phylogenetic and spatial distribution in natural ecosystems. These observations imply that most aquatic animals are exposed to these bacteria. Therefore, we screened
the viscera of various common aquatic animals for the
presence of STXs and TTX (Sato et al. 1993). HPLC
analyses and bioassays of the toxins partially purified
from the viscera showed that all species examined
showed the presence of both TTX and STXs, although
the level were very low (Table 4). For further confirmation, an extract of the chum salmon, Onchorhyncs keta,
in which low levels of TTX, STX, and neoSTX were detected by HPLC analysis, was prepared from a large
amount of the viscera (62 kg) of specimens collected at a
river mouth during the homing period. A toxin with a retention time identical to that of TTX was detected in the
HPLC analysis, but was lost during the purification
process; however, toxins corresponding to STX and
neoSTX were obtained in a highly pure state. Mass
spectrometry and NMR analyses clearly showed that
these toxins were STX and neoSTX (Sato et al. 1998).
These findings show that TTX and STXs are not substances found only in specific organisms but are widely
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 37. Electron micrograph of hepatocytes from Takifugu poecilonotus. (a) There are three hepatocytes in the electron
micrographs. Each cell contain many microorganism-like components (arrows). No abnormal bioreactions are seen in every
hepatocyte. F, fat droplet. Bar = 2 µm. (b) Enlarged part of (a). Three round microorganisms are seen. These are surrounded
by double membranes from the cytoplasms of the hepatocytes. Numerous ribosomes (R) and the thick septum (S) are demonstrable. Bar = 0.5 µm. Reprinted with permission from Kodama, Shimizu, Sato, Ogata and Terao. The infection of bacteria in
the liver cells of toxic puffer—A possible cause for organisms to be made toxic by tetrodotoxin in association with
bacteria. Harmful Marine Algal Blooms—Proceed. 6th Intern. Conference Toxic Marine Phytoplankton, Nantes, October
1993, P. Lassus et al., © Technnique et Documentation—Lavoisier, 1995.
Fig. 38. Double immunodiffusion of the extracts of the liver and isolated bacteria against antibody P and puffer liver. 1 and
4: the liver extract of a toxic specimen of the pufferfish Takifugu porphyreus; 2 and 6: the extract of bacteria isolated from the
liver, the concentration of the extracts is different; 3 and 5: the extract of antigenic bacteria to prepare antibody P. (a) Center:
antibody P, (b): Center the serum of puffer specimen T. porphyreus from which the liver (1 and 4) and bacteria (2 and 6) were
prepared. Antibody P was prepared by immunizing rabbit with homogenate of bacteria isolated from the liver of another toxic
specimen of T. porphyreus. Reprinted with permission from Kodama, Shimizu, Sato, Ogata and Terao. The infection of
bacteria in the liver cells of toxic puffer—A possible cause for organisms to be made toxic by tetrodotoxin in association
with bacteria. Harmful Marine Algal Blooms—Proceed. 6th Intern. Conference Toxic Marine Phytoplankton, Nantes, October 1993, P. Lassus et al., © Technnique et Documentation—Lavoisier, 1995.
doi:10.5047/absm.2010.00301.0001
© 2010 TERRAPUB, Tokyo. All rights reserved.
31
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
32
Table 4. Occurrence of TTX and STXs in common aquatic animals.
Species
Shark
Lemna ditropis
Part
TTX
Toxin composition (mg mL–1 solution*)
GTX1,4
GTX2,3
2.3
0.3
2.3
0.5
neoSTX
STX
Int
Liv
2.3
1.0
Int
Liv
1.1
0.7
Conger eel
Conger myriaster
Vis
1.3
Saury
Cololabis saira
Vis
1.0
Int
Liv
0.6
0.2
0.6
2.1
0.2
3.1
Int
Liv
1.9
1.6
0.9
0.1
0.7
0.4
0.2
0.1
Liv
1.7
Int
Liv
0.8
2.5
0.3
1.8
0.3
0.1
0.1
Crayfish
Procambarus clarkii
Hep
0.4
1.0
Rock crab
Hemigrapsus sanguineus
Hep
0.1
Rock crab
Pachygrapsus crassipes
Hep
0.1
Marine snail
Neptunea arthritica
Dig
Salmon
Oncorhynchus keta
Cod
Gadus macrocephalus
Parrotfish
Ypsiscarus ovifrons
Skipjack
Katuwonus pelamis
Filefish
Nevodom modestus
0.2
0.1
0.1
4.2
0.3
1.9
0.1
0.9
1.0
0.1
0.9
0.3
0.1
Int, intestine; Liv, liver; Vis, viscera; Hep, hepatopancreas; Dig, digestive gland.
*1 mL solution is equivalent to 100 g of organ.
This article was published in Toxic Phytoplankton Blooms in the Sea (Smayda and Shimizu, eds.), Sato, Ogata and Kodama,
Wide distribution of toxins with sodium channel blocking activity similar to tetrodotoxin and paralytic shellfish toxins in
marine animals, pp. 429–434, © Elsevier (1993).
distributed in common aquatic animals at low levels. As
shown in Table 4, no significant difference was observed
in the levels of these toxins in the viscera of various common animals, indicating that these toxins are not concentrated via the food web but rather they originate from
intestinal bacteria. More than 90% of the homing chum
salmon returning close to their home river do not consume any diet, and thus their stomachs are empty (Ueno
1993). All of the stomachs in the viscera used in the above
experiments were empty, thus the STXs found in the visdoi:10.5047/absm.2010.00301.0001
cera did not originate from the diet. These findings support the view that bacteria which produce TTX and STXs
are widely distributed in natural ecosystems.
15. Hypothesis on toxin accumulation in toxic organisms
As described above, bacteria which produce low
levels of toxins live in cells of the puffer and in
dinoflagellates which display high levels of TTX and/or
© 2010 TERRAPUB, Tokyo. All rights reserved.
M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010
Fig. 39. Hypothesis on the mechanism by which toxic organisms become toxic in association with infectious bacteria.
STXs. Toxic shellfish also possess these bacteria in their
cells, carried by the vector dinoflagellates. These bacteria grow in cells of the host organisms. The host organisms kill and digest some of the bacteria that have proliferated within the host cells and thereby maintain a suitable number of bacterial cells for their survival. That is,
direct biochemical interactions between the bacteria and
the host occur within the cells in the host organisms. The
finding that the number of bacteria found in the host is
few tends to support this idea. Based on these findings,
the hypothesis shown in Fig. 39 is proposed, suggesting
that TTX and STXs are not the final products of their
metabolisms but are intermediate products synthesized
by some bacterial substance which seems to be important for their survival. Recently, the biosynthetic pathway of STX proposed by Shimizu et al. (1984) has been
confirmed with STX biosynthesis gene and biosynthetic
intermediate in PSP toxins-producing cyanobacteria
(Kellmann et al. 2008a, b). However, occurrence of these
enzymes have not been reported in toxic dinoflagellates
such as Alexandrium species. Probably, the STX synthesized by bacteria is incorporated to “unknown biomolecule” suggested in Fig. 39. When the bacteria with
this unknown substance invade the toxic organisms such
as dinoflagellates and puffer, a part of bacteria is digested
by the defense mechanism of the host organisms. During
digestion, STXs or TTX bound with the biomolecule are
considered to be released.
Previously, we reported that TTX and STXs are released from a nontoxic high molecular weight fraction
prepared from toxic scallop digestive glands or toxic
puffer livers on digestion with RNase (Kodama et al.
1982b, 1983b). These findings suggest that RNA of toxinproducing bacteria is a possible candidate of the “unknown substance” shown in Fig. 39. This idea is consistent with the observation that the toxin content of the bacteria is very low (Kodama et al. 1990b), and that toxin
doi:10.5047/absm.2010.00301.0001
33
production by the bacteria increases significantly under
P-limiting conditions (Doucette and Trick 1995) as well
as under starved conditions (Kodama et al. 1990b). Toxic
animals and dinoflagellates are hosts which possess the
enzyme(s) required to digest the “unknown substance”
and release the toxins. If the “unknown substance” in
question is RNA, the enzyme required to digest this substance would be an RNase, an enzyme which occurs
widely in living organisms. Toxic organisms might
possess a specific type of RNase capable of digesting
foreign RNA which contains unusual components, STXs,
and/or TTX. Some strains of toxic dinoflagellates are
known to be nontoxic. The RNase in these strains might
lack the ability to digest foreign RNA containing unusual
components which are not essential for their growth.
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