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Toxins Produced by Benthic Dinoflagellates

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Takeshi Yasumoto
Takeshi Yasumoto
Takeshi Yasumoto
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Naoko Seino
Naoko Seino
Naoko Seino
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Yasutaka Murakami
Yasutaka Murakami
Yasutaka Murakami
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Michio Murata at Osaka University
Michio Murata
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Abstract

Nine species of benthic dinoflagellates collected in subtropical waters were cul tured, extracted,and tested for mouse lethality, ichthyotoxicity, and hemolytic activ ity. Hemolytic activity was detectable in all species, but the activities of Amphidinium carteri, A. k!ebsi, and Gambierdiscus toxicus were outstanding. G. toxicus showed the most potent mouse lethality. Two hemolytic constituents ofA. carteriwere deter mined to be mono- and di-galactoglycerolipids.Maitotoxin, producedby G. toxicus, was suggested to have a molecular weight of 3402 ±2 (m/z). Two potent toxins against mice were isolated from Prorocentrum !ima and identified as okadaic acid and 5-methylene-6-hydroxy-2-hexen-l-okadaate.
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Reference:Biol. Bull. 172: 128—131.(February,1987)
TOXINS PRODUCED BY BENTHIC DINOFLAGELLATES
TAKESHI YASUMOTO, NAOKO SEINO**, YASUTAKA MURAKAMI***,
AND MICHIO MURATA
Faculty ofAgriculture, Tohoku University, 1-1 Tsutsumidori.Amamiyamachi. Sendai 980. Japan
ABSTRACT
Nine species of benthic dinoflagellates collected in subtropical waters were cul
tured, extracted, and tested for mouse lethality, ichthyotoxicity, and hemolytic activ
ity. Hemolytic activity was detectable in all species, but the activities of Amphidinium
carteri, A. k!ebsi, and Gambierdiscus toxicus were outstanding. G. toxicus showed
the most potent mouse lethality. Two hemolytic constituents ofA. carteriwere deter
mined to be mono- and di-galactoglycerolipids. Maitotoxin, produced by G. toxicus,
was suggested to have a molecular weight of 3402 ±2 (m/z). Two potent toxins
against mice were isolated from Prorocentrum !ima and identified as okadaic acid
and 5-methylene-6-hydroxy-2-hexen-l-okadaate.
INTRODUCTION
Scientists first noticed the toxigenicity of benthic dinoflagellates when Gambier
discus toxicus was found to produce and to transmit ciguatoxin and maitotoxin to
herbivorous fish (Yasumoto et a!., 1977). Subsequent G. toxicus distribution surveys
revealed an abundance, in terms ofboth species and population, ofbenthic dinofla
gellates in coral reef communities. This observation suggests that toxic metabolites,
ifany, ofthese benthic species are taken up by herbivorous fish, as ciguatoxin is, and
contribute to the manifestation of the complex symptoms of ciguatera. The actual
occurrence of minor toxins in the viscera of herbivorous fish has been confirmed,
and the toxigenicity ofseveral benthic species has also been demonstrated previously
(Yasumoto et a!., 1976; Nakajima et a!., 1981). The present paperbriefly summarizes
our knowledge ofthe toxic benthic species and the chemical natures of their toxins;
ciguatoxin, however, has been described separately by Tachibana et a!. (1986).
MATERIALS AND METHODS
Benthic dinoflagellates collected at Okinawa, Japan, were cultured in a nutrient
enriched seawater medium described by Provasoli (1968). The following nine species
were tested forthe toxin production: Amphidinium carteri,A. kiebsi, Coo!ia monotis,
Gambierdiscus toxicus, Ostreopsis ovata, 0. siamensis, Prorocentrum concavum, P.
!ima, and P. rhathymum.
The harvested cells were extracted with boiling methanol. The methanol was re
moved by evaporation, and the residue was then suspended in water and extracted,
firstwith diethyl ether and then with 1-butanol. The residues obtained after evapora
*To whom correspondence should be addressed.
@ Present addresses: N. Seino, Ohmon Research Institute of Tobishi Pharm. Co. Ltd., 1-16-18
Ohmorinishi, Ohtaku, Tokyo 146,Japan; Y. Murakami, MITSUKAN Co. Ltd. 2-6 Nakamuracho, Handa,
Aichi Pref. 475, Japan.
128
ToxicigenicityofbenshicdinofiagellazesMouseHemolyticSpecieslethalityIchthyotoxicityactivityAmphidinium
carten+++++Amphidinium
klebsi++++++++Coolia
monotis——+Gambierdiscustoxicus+++++—+++++Ostreopsis
ovata+—+Ostreopsis
siamensis+++—+Prorocentrum
concavum++++++Prorocentrum
lima+++—+Prorocentrum
rhathymum——+
TOXINS OF BENTHIC DINOFLAGELLATES 129
TASLEI
The relativepotency is expressedby increasingthe number of +, the potency ofundetectable level is
expressed by —¿.
tion ofthe solvents were tested, respectively, for mouse lethality, ichthyotoxicity, and
hemolytic activity, as described previously (Nakajima et a!., 1981).
Three toxins, tentatively named PL toxin-i, -2, and -3, were isolated from P.
!ima by successive treatments on columns of silicic acid (CHC13-MeOH, stepwise),
Sephadex LH-20 (CHC13-MeOH 2:1), LiChroprep RP-2 (Merck, MeOH-H2O 2:1),
and ODS (Kyowaseimitsu, MeOH-H2O 4:1).
Five hemolytic compounds (hemolysin-i to -5) were present in A. carteri. Hemo
lysin-1 and -2 were purified on columns ofsilicic acid (CHC13-MeOH,stepwise) and
ODS Q-3 (Fujigel, MeOH-H2O 9:1). Hemolysin-3 to -5 were also purified in a similar
manner but further purification on a Toyopearl 40 column was necessary (MeOH
H2O, stepwise).
Purification of maitotoxin was carried out on columns of silicic acid (CHCI3-
MeOH, stepwise), Develosil ODS (Nomurakagaku, MeOH-H20, stepwise), and Dc
velosil TMS (MeCN-H20 35:65).
‘¿HNMR and ‘¿3CNMR spectra were taken on either a Nicolet NT 360 spectrome
ter or a JEOL FX-100 spectrometer, and mass spectra were taken on a Hitachi M-80
mass spectrometer.
RESULTS
Bioassays indicate that all the dinoflagellates are toxic by at least one ofthe assay
methods, as shown in Table I. Mouse lethality was observed in A. carteri, A. kiebsi,
G. toxicus, 0. siamensis, P. concavum, and P. lima. The toxicity of G. toxicus to the
mouse was outstanding. Hemolytic activity was most prominent in A. carteri and A.
kiebsi, although observed in all species tested. Potent ichthyotoxicity was observed
in A. carteri, A. kiebsi, and P. concavum.
The chromatographic and spectral analyses of PL toxin-2, the major toxin pro
duced by P. !ima, proved it to be okadaic acid (Murakami et a!., 1982), a cytotoxic
polyether fatty acid derivative previously isolated from sponges (Tachibana et a!.,
1981). PL toxin-i was found to be a mixture ofdiol esters ofokadaic acid. The struc
tures of okadaic acid and the major ester in the PL toxin-i fraction are shown in
Figure 1. PL toxin-3 was shown to be a tertiary amine having a molecular weight of
981 (m/z). Elucidation ofits chemical structure is under way.
130 T. YASUMOTO ET AL
I: R = H
II: R =
FIGURE1. Okadaicacid(I)and one ifits diolesters(II) fromProrocentrumlimo.
Among the five hemolysins ofA. carteri, hemolysin-l and -2 were more abundant
than the other three. The structures of hemolysin-1 and -2 were determined to be
O-fi-D-galactopyranosyl-(i-l)-3-O-octadecatetraenoyl-D-glycerol and O-a-D-galac
tosyl-(i-6)-O-@-D-galactopyranosyl-(i-1)-3-O-octadecatetraenoyl-D-glycerol, respec
tively (Fig. 2). The hemolytic activities ofhemolysin-i and -2 were 80%and 25% of
that of the commercial sapomn (Merck), respectively. The hemolytic activities of
hemolysin-3, -4, and -5 were 100, 9, and 2 times more potent than the commercial
saponin, respectively. Theirchemical structures were indicated to be entirely different
from those ofhemolysin-i and -2. Further structural work is under way.
Maitotoxin judged to be homogeneous by HPLC and TLC was obtained as an
amorphous solid. It was extremely lethal to mice (0.13 pg/kg, ip). Mass spectra sug
gested the molecular weight of 3402 ±2 (m/z). Chemical and spectral analyses mdi
cated the absence of any amino acid or fatty acid moieties in the molecule. Further
analyses ofthe structure are underway.
Disci@SSIoN
The occurrence of diverse toxins in benthic dinoflagellates was confirmed. Out of
the nine species tested, six produced mouse-lethal toxins, three ichthyotoxins, and all
species produced hemolytic substances. Such a high occurrence of toxins is a charac
HO HO
HO 0 HemolysinI
HO OH
HO 0
‘¿‘@/@‘
HemolysinII
FIGURE 2. Hemolysin land H from Amphidinium carteri.
TOXINSOF BENTHICDINOFLAGELLATES 131
teristic feature of benthic dinoflagellates. The biological and ecological significance
of the toxins are not clear at present. Whether the toxins deter the growth of other
microorganisms and thus benefit the elaborators remains to be tested.
The monoacylgalactolipids (hemolysin-i and -2) are closely related to the known
intermediate metabolites ofphotosynthesis, and therefore are likely to be widely dis
tributed. The compounds may not be involved in ciguatera, but they could be respon
sible, in part, for fish kills during blooms of dinoflagellate species with no known
ichthyotoxins.
Production ofokadaic acid by P. !ima is interesting because ofits chemical resem
blance to ciguatoxin and its potent diarrheagenicity. As P. lima is widely and densely
distributed in coral reefs, there is a possibifity that the compound, like ciguatoxin, is
taken up by herbivorous fish and thus contributes to the diarrhea which is frequently
seen in ciguatera patients.
The presence of maitotoxin in the viscera of surgeonfish has already been con
firmed, and the toxin has been suspected of contributing to the diverse ciguatera
symptoms seen in patients who have eaten herbivorous fish without first eliminating
the viscera. The most characteristic feature ofmaitotoxin is its high lethality to mice
(0. 13 @tg/kg,ip), which is 70 times that of saxitoxin or tetrodotoxin. The specific ac
tion ofmaitotoxin, to enhance the calcium ion influx through excitable membranes,
was first discovered by Ohizumi's group (Takahashi et a!., 1982; Ohizumi et aL,
1983). Today the compound is being used extensively as a chemical tool in biomedi
cal research.
ACKNOWLEDGMENTS
The authors aregrateful to Dr. I. Nojima ofJOEL Co. and Drs. H. Naoki and T.
Iwashita of the Suntory Institute for Bioorganic Research for the spectral measure
ments. This work was supported in part by Grant-in-Aid from the Ministry of Educa
tion, Culture, and Science, Japan.
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  • HARMFUL ALGAE
Epibenthic dinoflagellates occur globally and include many toxin-producing species of concern to human health and benthic ecosystem function. Such benthic harmful algal blooms (BHABs) have been well described from tropical and sub-tropical coastal environments, but assessments from north temperate waters, e.g., northern Europe, and polar regions are scarce. The present study addressed the biodiversity and distribution of potentially toxic epibenthic dinoflagellate populations along the west coast of Sweden (Kattegat-Skagerrak) by morphological and molecular criteria. Morphological analysis conducted by light- and electron-microscopy was then linked by DNA barcoding of the V4 region of 18S rRNA gene sequences to interpret taxonomic and phylogenetic relationships. The presence of two potentially toxigenic epibenthic dinoflagellates, Prorocentrum lima (Ehrenberg) F.Stein and Coolia monotis Meunier was confirmed, along with a description of their spatial and temporal distribution. For P. lima, one third of the cell abundance values exceeded official alarm thresholds for potentially toxic BHAB events (>1000 cells gr–1 of macroalgae fresh weight). The same species were recorded consecutively for two summers, but without significant temporal variation in cell densities. SEM analyses confirmed the presence of other benthic Prorocentrum species: P. fukuyoi complex, P. cf. foraminosum and P. cf. hoffmannianum. Analyses of the V4 region of the 18S rRNA gene also indicated the presence P. compressum, P. hoffmannianum, P. foraminosum, P. fukuyoi, and P. nanum. These findings provide the first biogeographical evidence of toxigenic benthic dinoflagellates along the west coast of Sweden, in the absence of ongoing monitoring to include epibenthic dinoflagellates. Harmful events due to the presence of Coolia at shellfish aquaculture sites along the Kattegat-Skagerrak are likely to be rather marginal because C. monotis is not known to be toxigenic. In any case, as a preliminary assessment, the results highlight the risk of diarrhetic shellfish poisoning (DSP) events caused by P. lima, which may affect the development and sustainability of shellfish aquaculture in the region.
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本邦における底生性有毒渦鞭毛藻に関する研究の現状_藻類 [Current status of benthic toxic dinoflagellate research in Japan_The Japanese Journal of Phycology (Sôrui)]
Article
Full-text available
  • Jul 2022
Several species of three benthic dinoflagellate genera (Gambierdiscus, Fukuyoa, and Prorocentrum) produce toxins causing ciguatera poisoning or diarrhetic shellfish poisoning. This review article summarises the current status of research on these genera in Japan. The species diversity is high, as eleven Gambierdiscus, one Fukuyoa, and nine Prorocentrum species/phylotypes have been reported. These species/phylotypes show a unique distribution pattern at sites from surface to 30 m depths in the subboreal, temperate, and/or subtropical zones. Gambierdiscus cell densities in summer/autumn are higher than those in spring/winter in the temperate zone. Prorocentrum cell densities in the subtropical zone are higher than those in the temperate zone. Two Gambierdiscus species produce maitotoxin-1 and/or 44-methylgambierone and four Prorocentrum species/phylotypes produce okadaic acid and/or dinophysistoxin-1. Several species/phylotypes of these two genera show toxicity to animals. Five Gambierdiscus and one Prorocentrum species/phylotypes show a unique growth characteristic in various temperature, salinity, or light intensity conditions.
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Maitotoxin, a Ca2+ channel activator candidate
Article
Full-text available
  • Aug 1982
Effects of maitotoxin, the most potent marine toxin, were studied using a rat pheochromocytoma cell line, PC12h. A low concentration (10(-8) g/ml) of maitotoxin induced a profound increase in CA2+ influx into PC12h cells and the Ca2+-dependent release of [3H]norepinephrine from them. The effects of maitotoxin were not affected by treatment with tetrodotoxin (10(-6) M) and were observed even in the absence of external Na+. Furthermore, these effects were markedly inhibited or abolished by treatment with verapamil (30-300 microM), Mn2+ (5 mM), or tetracaine (1 mM). These results suggest that maitotoxin activates the voltage-dependent calcium channels of PC12h cells.
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