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Species specificity and potential roles of Karlodinium micrum toxin

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Karlodinium micrum is a toxic mixotrophic dinoflagellate that has been responsible for fish kills in coastal environments worldwide. The role that karlotoxins play in the life history of K. micrum is unknown, but may contribute to its bloom-forming ability. We tested the hypothesis that karlotoxins could inhibit the growth of other protists depending on the sterol composition of target cell membranes. We also examined the effect of toxin addition on feeding rates of K. micrum on a flagellated prey, Storeatula major. Dose-dependent effects of isolated karlotoxin (KmTX2) were tested in growth bioassays (24-48h) of K. micrum, three raphidophytes (Heterosigma akashiwo, Fibrocapsa japonica and Chattonella subsalsa), two cryptophytes (S. major andPyrenomonas salina), and the dinoflagellates Amphidinium carterae, Pfiesteria piscicida andP. shumwayae. Growth of K. micrum, P. salina, A. carterae and P. piscicida were not affected by karlotoxin additions up to 1 000ng ml−1. Other organisms showed growth inhibition at concentrations between 500ng ml−1 and 1 000ng ml−1. Predation by K. micrum on S. major was significantly higher in the presence of 25ng ml−1 KmTX2. The results are consistent with a role for karlotoxin in allelopathic inhibition of competitors and/or prey immobilisation depending on sterol composition.
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African Journal of Marine Science 2006, 28(2): 415–419
Printed in South Africa — All rights reserved
Copyright © NISC Pty Ltd
AFRICAN JOURNAL OF
MARINE SCIENCE
EISSN 1814–2338
Species specificity and potential roles of Karlodinium micrum toxin
JE Adolf1*, TR Bachvaroff1, DN Krupatkina1,H Nonogaki1, PJP Brown2, AJ Lewitus2,3, HR Harvey4and AR Place1
1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA
2Belle W Baruch Institute for Marine and Coastal Sciences, University of South Carolina, Hollings Marine Laboratory, 331
Fort Johnson Road, Charleston, SC 29412, USA
3Marine Resources Research Institute, South Carolina Department of Natural Resources, Hollings Marine Laboratory, 331
Fort Johnson Road, Charleston, SC 29412, USA
4Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, Solomons, MD 20688, USA
* Corresponding author, e-mail: adolf@umbi.umd.edu
Introduction
Karlodinium micrum is a cosmopolitan toxic dinoflagellate
that has been associated with fish kills along the eastern
seaboard of the United States and in other temperate
coastal environments (Deeds et al. 2002, Kempton et al.
2002). It produces compounds, putatively named karlo-
toxins, that have been shown to be haemolytic, cytotoxic
and ichthyotoxic in laboratory experiments (Deeds et al.
2002, Kempton et al. 2002).
The ecological function that karlotoxins serve for K. micrum
is unknown. Particularly for dinoflagellates, production of
cytotoxic metabolites may yield a competitive advantage for
the producing organism that enables it to dominate the
phytoplankton and form blooms. Dinoflagellates, including K.
micrum, have relatively slow autotrophic growth rates and
thus may benefit significantly from alternative strategies that
allow them to out-compete other phytoplankton. Allelopathy,
the production of substances that hinder the growth or
physiological function of potentially competitive organisms, is
one such strategy (Legrand et al. 2003).
Mixotrophic nutrition is another means by which K. micrum
may out-compete other phytoplankton: K. micrum can feed on
other protists and achieve mixotrophic growth rates two- to
three-fold higher than its maximum autotrophic growth rate
(Li et al. 1999). Recent work with the prymnesiophyte
Prymnesium parvum (Skovgaard and Hansen 2003) has
suggested that toxin production can immobilise and facili-
tate capture of motile prey. The role of toxin production in
feeding by K. micrum has not been investigated.
Here, we tested the species specificity and potential roles
of karlotoxins produced by K. micrum. We hypothesised
that the lysis of different target species by karlotoxin would
depend on sterol composition of the target cell’s membrane
(see Deeds and Place 2006). Because laboratory cultures
and natural populations of K. micrum have been observed
feeding on flagellated cryptophyte prey (Li et al. 1999,
2000), we also hypothesised that pre-treatment of prey with
karlotoxin would result in higher feeding rates as a result of
prey immobilisation.
Material and Methods
Cultures
The cultures used in these experiments are listed in Table 1.
Karlodinium micrum is a toxic mixotrophic dinoflagel-
late that has been responsible for fish kills in coastal
environments worldwide. The role that karlotoxins play
in the life history of K. micrum is unknown, but may
contribute to its bloom-forming ability. We tested the
hypothesis that karlotoxins could inhibit the growth of
other protists depending on the sterol composition of
target cell membranes. We also examined the effect of
toxin addition on feeding rates of K. micrum on a flagel-
lated prey, Storeatula major. Dose-dependent effects of
isolated karlotoxin (KmTX2) were tested in growth
bioassays (24–48h) of K. micrum, three raphidophytes
(Heterosigma akashiwo, Fibrocapsa japonica and
Chattonella subsalsa), two cryptophytes (S. major and
Pyrenomonas salina), and the dinoflagellates Amphi-
dinium carterae, Pfiesteria piscicida and P. shumwayae.
Growth of K. micrum, P. salina, A. carterae and P. pisci-
cida were not affected by karlotoxin additions up to
1 000ng ml–1. Other organisms showed growth inhibi-
tion at concentrations between 500ng ml–1 and 1 000ng
ml–1. Predation by K. micrum on S. major was signifi-
cantly higher in the presence of 25ng ml–1 KmTX2. The
results are consistent with a role for karlotoxin in
allelopathic inhibition of competitors and/or prey immo-
bilisation depending on sterol composition.
Keywords: allelopathy, Karlodinium, karlotoxin, mixotrophy
Adolf, Bachvaroff, Krupatkina, Nonogaki, Brown, Lewitus, Harvey and Place416
Growth rate bioassays
Bioassays were conducted in three separate sets of
experiments. Purified karlotoxin, KmTX2, was added to
growth medium at twice the target treatment concentration
in sterile glass tubes. An equivalent volume of carrier
methanol was added to controls that did not receive toxin.
Equal volumes of algal culture and karlotoxin-containing
media were then pipetted, using glass pipettes, into wells
of a 24-well polystyrene well plate. In Experiment 1, run in
triplicate, cells were enumerated under a microscope after
48h exposure. In Experiment 2, a series of target cell
concentrations (3 250 cells ml–1, 7 250 cells ml–1, 75 000
cells ml–1 and 150 000 cells ml–1) was used and cells were
enumerated by a Coulter®Counter after 20h exposure. In
Experiment 3, cells were enumerated under the micro-
scope after 24h exposure to toxin. The cell count data for
each karlotoxin concentration tested in each experiment
were expressed as a growth rate,
where N0and N1are the initial and final target cell concen-
trations respectively (cells ml–1), and t0and t1are the start
and end times respectively (days). Growth rate was plotted
against karlotoxin concentration and subjected to a linear
regression analysis (S-PLUS Pro 6.1) to determine whether
karlotoxin concentration had a significant effect on growth
rate. In Experiments 1 and 3, where each karlotoxin
concentration was replicated, all data were used in fitting
the linear regressions.
Feeding experiments
Cultures of K. micrum strains CCMP 1974 (34 110 cells
ml–1) and CCMP 2064 (87 865 cells ml–1) grown in f/2-Si
medium were diluted to equal cell concentrations (34 000
cells ml–1) with growth medium. S. major prey (334 300
cells ml–1) were diluted three-fold with f/2-Si medium
containing a 10% P concentration. This diluted prey
suspension was used to prepare a double karlotoxin treat-
ment concentration of 50ng ml–1 KmTX2 in a sterile glass
tube. Treatments with no toxin but an equivalent volume
(0.03% v/v) of carrier methanol were prepared as controls.
Triplicate treatments were prepared in a polystyrene 24-well
plate by pipetting prey suspension into wells containing an
equal volume of either growth medium (f/2-Si), K. micrum
CCMP 1974 or K. micrum CCMP 2064. The well plate was
covered, wrapped with polyvinyl chloride film and returned
to the growth chamber. After 5h, the treatments were fixed
by adding gluteraldehyde (1% final concentration) and
allowed to settle for >24h at 4°C. At least 100 random cells
were counted at 400Xmagnification, under an Olympus
inverted microscope equipped with phase contrast, and
scored for the presence or absence of an orange inclusion
indicative of an ingested cryptophyte.
Sterol analyses
Sterol analyses were performed as described by Deeds
and Place (2006) and Place et al. (2006).
Results and Discussion
In Experiment 1, raphidophytes (with exception of H. aka-
shiwo 2) and the cryptophyte S. major were affected by
karlotoxin addition, resulting in a reduction of growth rate that
was dependent on toxin concentration (Figure 1, Table 2). K.
micrum was not affected by toxin addition (Figure 1, Table 2).
In Experiment 2, the dinoflagellate A. carterae and the
cryptophyte P. salina were not affected by karlotoxin
although the cryptophyte S. major was affected, except in
one of the cell density treatments (Table 2). P. shumwayae
was affected by karlotoxin addition whereas P. piscicida was
not (Table 2). P. shumwayae responded to toxin addition by
forming cysts, resulting in an apparent reduction of cell
numbers that was expressed as a reduction of growth rate
in the experiments. The sterol composition of these two
species of Pfiesteria differed in that, when compared with P.
piscicida, P. shumwayae showed elevated levels of brassi-
casterol, the presence of [[(3β, 22E)-ergosta-7,22-dien-3-
yl]oxy]trimethyl-sterols, and the absence of two 4α-methyl
sterols that were abundant in P. piscicida (Table 3).
Feeding experiments
Ingestion rates were 2–3 times higher on prey in the
presence of 25ng ml–1 karlotoxin (Figure 2a, b). Treatment
of prey with the double toxin concentration of 50ng ml–1 for
approximately 10 minutes before dilution at the beginning
of the feeding assay had no effect on prey cell numbers.
The production of bioactive compounds by phytoplankton
presumably yields an ecological advantage to the producer,
although this aspect of toxin production is far less under-
stood than the effects of toxins on human health or econo-
mic interests such as fisheries. Our interest in the biological
role(s) of karlotoxin stems from the desire to understand the
evolutionary origin of toxin production, and from a desire to
understand what factors help K. micrum form blooms in
nature. The present results show that high doses of
karlotoxin (>500ng m–1) inhibited the growth of raphido-
phytes, and one of two cryptophyte and Pfiesteria species
Table 1: Cultures used in the experiments
Maintenance
Culture Strain conditions
Chattonella subsalsa CAAE 1662X 20ppt f/2-Si
Fibrocapsa japonica C-101 20ppt f/2-Si
Heterosigma akashiwo CAAE 1663X 20ppt f/2-Si
Karlodinium micrum 010410-1C6 30ppt f/2-Si
K. micrum CCMP 1974 15ppt f/2-Si
K. micrum CCMP 2064 15ppt f/2-Si
Pyrenomonas salina 15ppt f/2-Si
Storeatula major Strain-g 15ppt f/2-Si
Pfiesteria piscicida CCMP 1829 15ppt
P. shumwayae CCMP 2089 15ppt
Growth rate day
N
N
tt
=
10
10
1
ln
African Journal of Marine Science 2006, 28(2): 415–419 417
tested. A. carterae and K. micrum were not affected by the
toxin. Furthermore, K. micrum ingested more cryptophytes
in the presence of a low dose (25ng ml–1) of karlotoxin in a
5h feeding assay.
Allelopathic chemicals are most effective when the
producing organism is not affected by the allelochemical.
K. micrum is immune to its own toxin (Deeds and Place
2006), and we suspect that specificity of karlotoxin
depends on the sterol composition of the target cell’s
membrane. Sterol composition is a critical factor determin-
ing toxicity in several pore-forming toxins (see Deeds and
Place 2006 for references). The sterol dominant in K.
micrum (gymnodinosterol [(24S)-4α-methyl-5α-ergosta-
8(14),22-dien-3β-ol]; Leblond and Chapman 2002) did not
interact strongly with karlotoxin (Deeds and Place 2006).
Ergosterol and cholesterol did interact strongly with karlo-
toxin, making membrane pore formation and cytotoxicity
more likely in organisms containing these membrane
sterols (Deeds and Place 2006). These observations point
to possible roles of the 4α-methyl group or the 8(14)
double bond (both present in gymnodinosterol, both absent
in cholesterol and ergosterol) in determining whether or not
a sterol interacts with karlotoxin. Our growth response
results were consistent with this association in eight of nine
species (P. salina was the exception, see below). The
sterol composition of A. carterae (immune to KmTX2) is
dominated by amphisterol that contains both the 4α-methyl
and 8(14) double bond (Withers et al. 1979, the present
data). P. piscicida (immune to KmTX2) sterols are domi-
nated by dinosterol that contains a 4α-methyl but no 8(14)
double bond. Raphidophytes, cryptophytes and P. shum-
wayae have desmethyl sterols as major membrane
components (Volkman 2003). In the analyses, desmethyl
Figure 1: Representative plot showing growth rate plotted as a
function of KmTX2 (karlotoxin) concentration for K. micrum, F.
japonica and S. major. Similar plots were used to derive the
statistics in Table 2
Table 2: Linear regression results for growth rate vs KmTX2 concentration in bioassay experiments. KmTX2 concentration ranged from 0ng
ml–1 to 1 000ng ml–1 in each experiment. t0indicates cell abundance at the start of the experiment. The y-intercept (intercept) corresponds to
the growth rate of the target species in the absence of toxin. A negative slope indicates a suppression of growth rate by toxin
Cell abundance
Target species Class (t0, cells ml–1) Intercept Slope r2p-value
Experiment 1
H. akashiwo 1 Raphidophyte 28 634 0.74 0.001 0.35 0.020*
H. akashiwo 2 Raphidophyte 26 132 0.80 0.0009 0.26 0.062*
F. japonica Raphidophyte 36 153 0.34 0.0027 0.71 <0.001*
C. subsalsa Raphidophyte 5 850 0.17 – 0.0011 0.43 0.015*
S. major Cryptophyte 42 024 0.73 0.014 0.68 < 0.001*
K. micrum Dinoflagellate 28 634 0.10 0.0001 0.08 0.310*
Experiment 2
A. carterae Dinoflagellate 3 250 0.85 0.0003 0.18 0.475*
7 500 0.67 0.0002 0.61 0.117*
75 000 0.42 < 0.0000 0.01 0.887*
150 000 0.44 0.0002 0.32 0.435*
P. salina Cryptophyte 3 250 1.41 0.0006 0.68 0.088*
7 500 0.20 0.0001 0.03 0.782*
75 000 0.88 0.0002 0.02 0.856*
150 000 0.95 0.0008 0.42 0.349*
S. major Cryptophyte 3 250 1.13 0.0011 0.92 0.009*
7 500 0.34 0.0008 0.50 0.182*
75 000 0.77 0.0034 0.90 0.049*
150 000 –1.03 0.0038 0.94 0.033*
Experiment 3
P. piscicida Dinoflagellate 39 375 0.74 0.0002 0.03 0.362*
P. shumwayae Dinoflagellate 9 813 0.28 0.0007 0.28 0.004*
* Significant regression (p < 0.05)
1
2
3
0
1
200 400 600 800
KARLOTOXIN (ng ml1)
GROWTH RATE (day1)
K. micrum
(not affected)
F. japonica
(affected)
S. major
(affected)
0
Adolf, Bachvaroff, Krupatkina, Nonogaki, Brown, Lewitus, Harvey and Place418
brassicasterol was the major component of both S. major
and P. salina (>90%). The analyses also showed a higher
abundance of desmethyl sterols in the karlotoxin-sensitive
species of P. shumwayae (Table 3). The lack of a negative
effect of karlotoxin on P. salina may indicate that secondary
mechanisms protect this organism from lysis. It is note-
worthy that the growth rate bioassays used in this study
would not detect sublethal effects of karlotoxin.
The toxin concentrations that were cytotoxic in growth
rate bioassays (>500ng ml–1) would require production by
K. micrum abundances >50 000–500 000 cells ml–1 assu-
ming a toxin quota between 1pg cell–1 and 10pg cell–1
(Deeds et al. 2004) and the release of all cellular toxin into
the surrounding medium. However, observations suggest
that >90% of karlotoxin is cell-associated, albeit loosely,
under normal culture conditions (Deeds et al. 2002). K.
micrum sometimes blooms to concentrations exceeding
50 000 cells ml–1, but generally exists at numbers well
below 10 000 cells ml–1 (Li et al. 2000, Goshorn et al.
2004). These observations suggest that cell-to-cell contact,
enabling delivery of toxin directly to the target cell mem-
brane, may be an important aspect of the allelopathic/prey
immobilisation mechanism in K. micrum. Further experi-
ments are needed to address this conjecture.
We suggest that the stimulation of prey ingestion in the
presence of a low dose of karlotoxin results from sub-lethal
effects of the toxin, resulting in prey immobilisation. When K.
micrum is feeding on a cryptophyte prey, S. major, the pro-
cess of phagocytosis begins with a prey capture sequence
that lasts approximately 30 seconds. During that time, K.
micrum appears to capture the cryptophyte with a narrow
appendage resembling a line (i.e. tow line) that extends
2–5µm between predator and prey (Li et al. 1999). S. major is
flagellated and approximately half the mass of K. micrum, and
during this initial phase of prey capture the two cells are
observed to struggle against each other, often ending in prey
escape (videos available at adolf@umbi.umd.edu). Our
tentative conclusion is that low doses of karlotoxin immobilise
S. major, reducing prey escape success resulting in higher
ingestion rates by K. micrum. Based on these findings, it
appears that karlotoxins may provide both a potential allelo-
pathic and prey capture advantage to K. micrum.
Acknowledgements — This work was funded in part by grants from
Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) and
National Institute of Environmental Health Sciences (to ARP), and
National Oceanic and Atmospheric Administration and Centers for
Disease Control and Prevention (to AJL). This is contribution no. 05-136
from the UMBI Center of Marine Biotechnology, no. 3993 from the
University of Maryland Center for Environmental Science, and no.136
from the ECOHAB programme.
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from the flame ionisation detector (FID) trace obtained by gas chromatography
Abundance (%)
Sterol P. shumwayae P. piscicida
(dinosterol) — 4α,23,24-trimethyl-5α-cholest-22-en-3β-ol 20.4 28.5
(brassicasterol) — 24-methylcholesta-5,22-dien-3β-ol 33.1 07.0
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4α,24-dimethylcholestan-3β-ol nd 12.2
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nd = Not detected
CONTROL 25ng ml1
KmTX2
INGESTED PREY PER K. micrum
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Prey: S. major (cryptophyte)
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p = 0.006
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(a)
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CCMP for (a) 2064 and (b) 1974
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... 1789). Karlodinium veneficum (original name: Gymnodinium veneficum; synonym: Karlodinium micrum, Gymnodinium micrum, Gyrodinium galatheanum, Woloszynskia micra, and Gyrodinium estuariale) is the type species and also the most intensively and extensively investigated one in the field and the laboratory [19][20][21][22][23]. Species in the genus of Karlodinium, such as K. veneficum, K. armiger, K. corsicum and K. aculat, have been reported to be associated with many toxic events and caused mortality of fishes, mussels and zooplanktons [18,[24][25][26][27][28][29][30]. ...
... During this stage, less than half of the prey cells (R. salina) were captured. Occasionally, a capture filament, an up to 10 µm long structure, which has also been reported in other phagocytosing dinoflagellates, such as Peridiniopsis berolinensis [63] and K. veneficum [19,20], was observed to attach the prey. When the capture succeeding, the predator placed its sulcal area, where the phagocytosis took place, facing the prey and revolved around its anterior-posterior axis. ...
... HABs of K. veneficum were assisted by karlotoxins and contributed to accumulations of toxic K. veneficum based on their relatively higher phagotrophic capacity compared to non-toxic cells. High densities of K. veneficum, when harmful blooms occurred, exhibited allelopathy to other co-occurred algae by suppressing their physiological activity and growth rates [19,88,120], which induced other microalgae species more favorable to being captured. ...
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... K. veneficum produces a suite of polyketide toxin congeners (karlotoxins) [11][12][13]. A series of toxicity tests on different isolates of K. veneficum have confirmed their hemolytic, ichthyotoxic, and cytotoxic properties and their toxic effect on the development of sea urchin embryos [7,[14][15][16]. K. veneficum is a constitutive mixotroph able to perform photosynthesis and to eat various prey by phagocytosis. ...
... K. veneficum is a constitutive mixotroph able to perform photosynthesis and to eat various prey by phagocytosis. Karlotoxins participate in predation or in grazer deterrence [3,14,17]. During predation, these toxins are produced and secreted into the water to immobilize the prey [18]. ...
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Biosurfactant has potential application value in the removal of microalgal blooms, but the ecological risks require more research. In this paper, the effects of surfactin on the toxic dinoflagellate Karlodinium veneficum were studied. The coaction of surfactin and K. veneficum was also evaluated through toxicological experiments on Artemia and juvenile clams. The results showed that: (1) in the concentration range of 0–10 mg/L, surfactin significantly killed algal cells in a dose-dependent manner within 48 h; the 24 h EC50 was 3.065 mg/L; (2) K. veneficum had the ability to restore population growth after stress reduction and the restored proliferation was positively correlated with the initial surfactin concentration; (3) the ability to restore population growth was associated with protection afforded by the promotion of antioxidant enzymes, including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), whose increase was positively correlated with the surfactin concentration; (4) the toxicity of the coculture of surfactin and K. veneficum was significantly greater than that of the K. veneficum culture or surfactin alone and was dose and time dependent. The potential ecological risks should be considered when applying biosurfactants, such as surfactin, in the removal of harmful algal blooms.
... Results of this study point toward potential impacts of climate change on trophic transfer efficiency by K. veneficum as both predator and as prey [42,79]. Increased toxicity of K. veneficum as would be expected under elevated CO 2 [42], for example, may reduce top down control on this species by meso-and microzooplankton predators [41, 80] while increasing predation by K. veneficum on other protists [38,79]. ...
... Results of this study point toward potential impacts of climate change on trophic transfer efficiency by K. veneficum as both predator and as prey [42,79]. Increased toxicity of K. veneficum as would be expected under elevated CO 2 [42], for example, may reduce top down control on this species by meso-and microzooplankton predators [41, 80] while increasing predation by K. veneficum on other protists [38,79]. For example, mixed prey experiments described in Adolf et al. [41] showed that the presence of toxic K. veneficum inhibited grazing by the heterotrophic dinoflagellate, Oxyrrhis marina, on co-occurring non-toxic strains. ...
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There is little information on the impacts of climate change on resource partitioning for mixotrophic phytoplankton. Here, we investigated the hypothesis that light interacts with temperature and CO2 to affect changes in growth and cellular carbon and nitrogen content of the mixotrophic dinoflagellate, Karlodinium veneficum, with increasing cellular carbon and nitrogen content under low light conditions and increased growth under high light conditions. Using a multifactorial design, the interactive effects of light, temperature and CO2 were investigated on K. veneficum at ambient temperature and CO2 levels (25°C, 375 ppm), high temperature (30°C, 375 ppm CO2), high CO2 (30°C, 750 ppm CO2), or a combination of both high temperature and CO2 (30°C, 750 ppm CO2) at low light intensities (LL: 70 μmol photons m-2 s-2) and light-saturated conditions (HL: 140 μmol photons m-2 s-2). Results revealed significant interactions between light and temperature for all parameters. Growth rates were not significantly different among LL treatments, but increased significantly with temperature or a combination of elevated temperature and CO2 under HL compared to ambient conditions. Particulate carbon and nitrogen content increased in response to temperature or a combination of elevated temperature and CO2 under LL conditions, but significantly decreased in HL cultures exposed to elevated temperature and/or CO2 compared to ambient conditions at HL. Significant increases in C:N ratios were observed only in the combined treatment under LL, suggesting a synergistic effect of temperature and CO2 on carbon assimilation, while increases in C:N under HL were driven only by an increase in CO2. Results indicate light-driven variations in growth and nutrient acquisition strategies for K. veneficum that may benefit this species under anticipated climate change conditions (elevated light, temperature and pCO2) while also affecting trophic transfer efficiency during blooms of this species.
... Investigations of chemical interference between microalgae species have focused on effects of toxin-producing, Harmful Algal Bloom (HAB) species on other toxic and non-toxic microalgae [2]. The bulk of these studies has reported growth inhibition on target microalgae linked to cell lysis, shifts in respiration, protein synthesis and gene expression [9][10][11][12][13][14][15][16][17]. However, positive allelopathy has also been reported by a limited number of studies [15,[18][19][20]. ...
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Highlights •We show that microalgae demonstrate a range of responses to foreign exometabolites. •Biomass suppression caused by Tetraselmis was linked to L-histidinal. •Biomass enhancement was only observed in Heterosigma akashiwo. •Luxurious P uptake can also occur as a response to foreign species exometabolites. •Tiliacorine is reported for the first time from the mixotroph H. akashiwo.
... To better understand Alexandrium BECs activity, it is instructive to compile and compare knowledge about other toxins of microalgal origin known to be involved in the several toxic and lytic activities. Such compounds are karlotoxins produced by Karlodinium veneficum [257][258][259][260][261][262], karmitoxin produced by Karlodinium armiger Berholtz, Daugbjerg and Moestrup [263], amphidinols produced by Amphidinium spp. [264,265], or prymnesins produced by the haptophyte Prymnesium parvum N.Carter [266,267]. ...
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Various species of Alexandrium can produce a number of bioactive compounds, e.g., paralytic shellfish toxins (PSTs), spirolides, gymnodimines, goniodomins, and also uncharacterised bioactive extracellular compounds (BECs). The latter metabolites are released into the environment and affect a large range of organisms (from protists to fishes and mammalian cell lines). These compounds mediate allelochemical interactions, have anti-grazing and anti-parasitic activities, and have a potentially strong structuring role for the dynamic of Alexandrium blooms. In many studies evaluating the effects of Alexandrium on marine organisms, only the classical toxins were reported and the involvement of BECs was not considered. A lack of information on the presence/absence of BECs in experimental strains is likely the cause of contrasting results in the literature that render impossible a distinction between PSTs and BECs effects. We review the knowledge on Alexandrium BEC, (i.e., producing species, target cells, physiological effects, detection methods and molecular candidates). Overall, we highlight the need to identify the nature of Alexandrium BECs and urge further research on the chemical interactions according to their ecological importance in the planktonic chemical warfare and due to their potential collateral damage to a wide range of organisms.
... As membranes are amongst the first targets for A. minutum allelochemicals, it can be hypothesized that their biochemical composition might drive, at least partially, the sensitivity of species to A. minutum. The composition of membranes was already suggested to be an important factor in some allelochemical interactions (Adolf et al., 2006;Deeds and Place, 2006;Morsy et al., 2008aMorsy et al., , 2008bMa et al., 2011;Waters et al., 2015). ...
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Allelopathy is an efficient strategy by which some microalgae can outcompete other species. Allelochemicals from the toxic dinoflagellate Alexandrium minutum have deleterious effects on diatoms, inhibiting metabolism and photosynthesis and therefore give a competitive advantage to the dinoflagellate. The precise mechanisms of allelochemical interactions and the molecular target of allelochemicals remain however unknown. To understand the mechanisms, the short-term effects of A. minutum allelochemicals on the physiology of the diatom Chaetoceros muelleri were investigated. The effects of a culture filtrate were measured on the diatom cytoplasmic membrane integrity (polarity and permeability) using flow-cytometry and on the photosynthetic performance using fluo-rescence and absorption spectroscopy. Within 10 min, the unknown allelochemicals induced a depolarization of the cytoplasmic membranes and an impairment of photosynthesis through the inhibition of the plastoquinone-mediated electron transfer between photosystem II and cytochrome b 6 f. At longer time of exposure, the cyto-plasmic membranes were permeable and the integrity of photosystems I, II and cytochrome b 6 f was compromised. Our demonstration of the essential role of membranes in this allelochemical interaction provides new insights for the elucidation of the nature of the allelochemicals. The relationship between cytoplasmic membranes and the inhibition of the photosynthetic electron transfer remains however unclear and warrants further investigation.
... In the eukaryotic subset of the community, dinoflagellates comprised 69 ± 11% of transcriptomic read counts and 41 ± 11% of protein spectral counts with the majority of these proteins associated with the Kareniaceae family and specifically, Karlodinium and Karenia-like genera ( Fig. 1b and Extended Data Fig. 2). This family contains gymnodinoid dinoflagellates known to contain mixotrophic members [27][28][29][30] . While there has been concern that sequencing-based approaches may overestimate dinoflagellate populations due to their large genome size and tendency to post-transcriptionally regulate gene expression (Supplementary Information), the metaproteomic dataset provides 147 plotted using Ocean Data View (odv.awi.de). ...
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Marine microeukaryotes play a fundamental role in biogeochemical cycling through the transfer of energy to higher trophic levels and vertical carbon transport. Despite their global importance, microeukaryote physiology, nutrient metabolism and contributions to carbon cycling across offshore ecosystems are poorly characterized. Here, we observed the prevalence of dinoflagellates along a 4,600-km meridional transect extending across the central Pacific Ocean, where oligotrophic gyres meet equatorial upwelling waters rich in macronutrients yet low in dissolved iron. A combined multi-omics and geochemical analysis provided a window into dinoflagellate metabolism across the transect, indicating a continuous taxonomic dinoflagellate community that shifted its functional transcriptome and proteome as it extended from the euphotic to the mesopelagic zone. In euphotic waters, multi-omics data suggested that a combination of trophic modes were utilized, while mesopelagic metabolism was marked by cytoskeletal investments and nutrient recycling. Rearrangement in nutrient metabolism was evident in response to variable nitrogen and iron regimes across the gradient, with no associated change in community assemblage. Total dinoflagellate proteins scaled with particulate carbon export, with both elevated in equatorial waters, suggesting a link between dinoflagellate abundance and total carbon flux. Dinoflagellates employ numerous metabolic strategies that enable broad occupation of central Pacific ecosystems and play a dual role in carbon transformation through both photosynthetic fixation in the euphotic zone and remineralization in the mesopelagic zone.
... P. minimum, Velikova and Larsen 1999) and Karlodinium veneficum are common in the Chesapeake Bay (Li et al., 2000;Glibert et al., 2007;Marshall and Egerton 2009). The mixotrophic dinoflagellate K. veneficum (Li et al., 1999;Adolf et al., 2006a) produces karlotoxins (KmTxs), a class of bioactive compounds with hemolytic, cytolytic, ichthyotoxic, and allelopathic effects Kempton et al., 2002;Adolf et al., 2006b;Place et al., 2012;Dorantes-Aranda et al., 2015;Yang et al., 2019). This species is perhaps best known for causing finfish kills (Goshorn et al., 2004); however, it has also been shown to have harmful effects on zooplankton (Adolf et al., 2007;Waggett et al., 2008;Yang et al., 2019) and shellfish, including, blue mussels (Mytilus edulis, Nielsen and Strømgren 1991;Galimany et al., 2008), hard clams (Mercenaria mercenaria, Place et al., 2008), and some life stages of oysters (Crassostrea virginica and C. ariakensis, Glibert et al., 2007;Brownlee et al., 2008;Place et al., 2008;Stoecker et al., 2008;Lin et al., 2017). ...
Article
Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.
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Allelopathy is widespread in marine, brackish, and freshwater habitats. Literature data indicate that allelopathy could offer a competitive advantage for some phytoplankton species by reducing the growth of competitors. It is also believed that allelopathy may affect species succession. Thus, allelopathy may play a role in the development of blooms. Over the past few decades, the world’s coastal waters have experienced increases in the numbers of cyanobacterial and microalgal blooming events. Understanding how allelopathy is implicated with other biological and environmental factors as a bloom-development mechanism is an important topic for future research. This review focuses on a taxonomic overview of allelopathic cyanobacteria and microalgae, the biological and environmental factors that affect allelochemical production, their role in ecological dynamics, and their physiological modes of action, as well as potential industrial applications of allelopathic compounds.
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The lipophilic toxins from Karlodinium micrum, KmTX, have negative effects on several co-occurring phytoplankton species, yet appear to have no effect on K. micrum itself. One of these compounds, KmTX2, has differing toxicity towards eukaryotic membranes with differing sterol compositions (vertebrate > fungal > dinoflagellate). It is shown that KmTX2 causes lysis in a co-occurring potential grazer Oxyrrhis marina while having no effect on K. micrum itself. K. micrum has a unique membrane sterol profile dominated by (24S)-4α-methyl-5α-ergosta-8(14),22-dien-3-ol (gymnodinosterol), whereas O. marina was shown to possess 5,22-cholestadien-24-methyl-3-ol (brassicasterol) and 5-cholesten-3-ol (cholesterol) as its major membrane sterols. In accord with toxicity data from whole cells containing these sterols, free sterols were found to inhibit haemolysis in the order cholesterol > ergosterol > gymnodinosterol. It appears that certain sterols can form stable complexes with the toxin molecule, thereby sequestering it away from erythrocyte membranes. It is concluded that K. micrum protects itself from the membrane-disrupting properties of its own toxins by possessing a membrane sterol that does not interact with these compounds.
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It is considered self-evident that chemical interactions are a component of competition in terrestrial systems, but they are largely unknown in aquatic systems. In this review, we propose that chemical interactions, specifically allelopathy, are an important part of phytoplankton competition. Allelopathy, as defined here, applies only to the inhibitory effects of secondary metabolites produced by one species on the growth or physiological function of another phytoplankton species. A number of approaches are used to study allelopathy, but there is no standard methodology available. One of the methods used is cross-culturing, in which the cell-free filtrate of a donor alga is added to the medium of the target species. Another is to study the effect of cell extracts of unknown constituents, isolated exudates or purified allelochemicals on the growth of other algal species. There is a clear lack of controlled field experiments because few allelochemicals have been identified. Molecular methods will be important in future to study the expression and regulation of allelochemicals. Most of the identified allelochemicals have been described for cyanobacteria but some known toxins of marine dinoflagellates and freshwater cyanobacteria also have an allelochemical effect. The mode of action of allelochemicals spans a wide range. The most common effect is to cause cell lysis, blistering, or growth inhibition. The factors that affect allelochemical production have not been studied much, although nutrient limitation, pH, and temperature appear to have an effect. The evolutionary aspects of allelopathy remain largely unknown, but we hypothesize that the producers of allelochemicals should gain a competitive advantage over other phytoplankton. Finally, we discuss the possibility of using allelochemicals to combat harmful algal blooms (HABs). Allelopathic agents are used for biological control in agriculture, e.g. green manures to control soil diseases in Australia, but they have not yet been applied in the context of HABs. We suggest that phytoplankton allelochemicals have the potential for management of HABs in localized areas.
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Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom-forming dinoflagellates, including the toxic species Karlodinium micrum. Strains of K. micrum produce cytotoxic compounds (KmTX) that render cell membranes permeable to a range of small ions and molecules, resulting in cell death through osmotic lysis. Membrane sterol composition appears to play a role in the sensitivity of different algal species to the membrane-disrupting effects of KmTX. This sterol specificity also appears to be responsible for the apparent immunity of K. micrum to its own toxins. K. micrum has a unique sterol profile, shared only by the congeneric dinoflagellates Karenia brevis and K. mikimotoi, dominated by (24S)-4α-methyl-5α-ergosta-8(14), 22-dien-3β-ol (72% by weight). This sterol has recently been named gymnodinosterol. Analysis of the sterol content in Amoebophrya sp. infecting K. micrum showed gymnodinosterol also to be dominant (62%). This was not simply a reflection of retaining host lipid content because K. micrum contains octadecapentaenoic acid (18:5n3), largely in galactolipids of the chloroplast, whereas Amoebophrya sp. contained little to no 18:5n3. By having a sterol content similar to its host, Amoebophrya sp. is able to avoid cell lysis caused by the cytotoxic compounds produced by the host.
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Gyrodinium galatheanum (Braarud) Taylor 1995 is a common bloom-forming, potentially toxic photosynthetic dinoflagellate in Chesapeake Bay, USA. Abundance of this dinoflagellate achieved densities >4 x 103 cells ml-1 in the mid- and upper Bay during late spring and early summer of 1995 and 1996. Ingestion of cryptophytes by this dinoflagellate was detected in most samples collected from the Bay. During late spring and early summer, mean number of ingested cryptophytes per G.galatheanum was as high as 0.46 for dinoflagellate populations located in surface waters of the mid- and upper Bay where dissolved inorganic phosphorus was low. Observations on the distribution of G.galatheanum in Chesapeake Bay show that populations of this dinoflagellate were usually restricted to waters with salinities ranging from 7 to 18 psu, seasonally progressed up the estuary, and usually co-occurred with cryptophytes. Correlation analysis indicates that abundance of G.galatheanum and incidence of feeding was negatively correlated with dissolved inorganic phosphorus, and that incidence of feeding was positively correlated with abundance of cryptophyte prey. These results indicate that G.galatheanum is an important component of the Chesapeake Bay phytoplankton during the spring and summer. Our results suggest that the phagotrophic capability possessed by this phototrophic dinoflagellate may contribute to its success in a varying-resource environment like Chesapeake Bay.
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Gyrodinium galatheanum is a photosynthetic, mixotrophic dinoflagellate that is capable of ingesting other protists, including cryptophytes. Ingestion of cryptophycean prey involves formation of a protrusion near the flagellar pores in the sulcus region of the dinoflagellate, by which prey are captured and phagocytized. In phototrophically growing G. galatheanum, a total of 12 chlorophylls and carotenoids are detected using HPLC pigment analysis. In G. galatheanum that had been fed cryptophycean prey for 41 h, traces of pigments that were derived from prey were found. This suggests that ingested prey were not fully digested or that some chloroplasts from prey were retained by the dinoflagellate. G, galatheanum cultured in nutrient-replete medium had net positive growth under phototrophic conditions (i.e, without addition of prey). It could not survive in prolonged darkness even with sufficient food supply, and thus is incapable of strictly heterotrophic growth. Under mixotrophic conditions (i.e. in the light with addition of a saturating concentration of prey), growth rates of G. galatheanum were 2- to 3-fold higher than under strictly phototrophic conditions at the same irradiances. Mixotrophically grown G. galatheanum had higher cellular chi a, cell volume, and cellular car bon content than cultures grown without particulate food. Phagotrophy also leads to enhanced photosynthetic performance of G. galatheanum due to increased photosynthetic capacity (P(max)(cell)), and/or by increased photosynthetic efficiency (alpha(cell)), particularly when the cells were grown under low light and/or nutrient-limited conditions. These results indicate that G, galatheanum is an obligately phototrophic species and that both photosynthesis and phagotrophy play significant roles in supporting the higher growth rates associated with mixotrophic than with strictly autotrophic growth.
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Gyrodinium galatheanum (Braarud) Taylor 1995 is a common bloom-forming, potentially toxic photosynthetic dinoflagellate in Chesapeake Bay, USA. Abundance of this dinoflagellate achieved densities >4 � 10 3 cells ml -1 in the mid- and upper Bay during late spring and early summer of 1995 and 1996. Ingestion of cryptophytes by this dinoflagellate was detected in most samples collected from the Bay. During late spring and early summer, mean number of ingested cryptophytes per G.galatheanum was as high as 0.46 for dinoflagellate populations located in surface waters of the mid- and upper Bay where dissolved inorganic phosphorus was low. Observations on the distribution of G.galatheanum in Chesapeake Bay show that populations of this dinoflagellate were usually restricted to waters with salinities ranging from 7 to 18 psu, seasonally progressed up the estuary, and usually co-occurred with cryptophytes. Correlation analysis indicates that abundance of G.galatheanum and incidence of feeding was negatively correlated with dissolved inorganic phos- phorus, and that incidence of feeding was positively correlated with abundance of cryptophyte prey. These results indicate that G.galatheanum is an important component of the Chesapeake Bay phyto- plankton during the spring and summer. Our results suggest that the phagotrophic capability possessed by this phototrophic dinoflagellate may contribute to its success in a varying-resource environment like Chesapeake Bay.
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Toxin production is widespread among aquatic microalgae, suggesting a functional advantage for organisms producing toxic compounds. However, the biological role of algal toxin production is only vaguely understood. Here, we show that excretion of a toxic substance in the phagotrophic phytoflagellate Prymnesium parvum(Prym- nesiophyceae) constitutes a mechanism to immobilize and seize motile prey. Feeding frequency of P. parvum in dilute batch cultures was low when fed the motile prey Heterocapsa rotundata (dinoflagellate). However, dense cultures caused immobilization of H. rotundata cells, thereby allowing P. parvum to feed on them. In contrast, when fed a nonmotile prey—the diatom Thalassiosira pseudonana—feeding frequency was high, even in dilute P. parvum cultures. We could demonstrate that feeding frequency of P. parvum on H. rotundata was positively cor- related with the measure of the toxic effect causing immobilization and lysis of prey cells. The fact that the toxic effect on H. rotundata was found in cell-free filtrate of P. parvum cultures suggests that immobilization and lysis of prey cells were caused by the excretion of toxins. Blooms of planktonic algae are common in aquatic envi- ronments, and harmful algal blooms cause substantial com- mercial problems for the exploitation of marine and fresh- water resources, as well as for recreational purposes (e.g., Hallegraeff 1993). The harmful effects of algal blooms are typically from toxins produced by the algae. The question of why algae produce toxins has, therefore, been a point of much speculation, but few facts exist on possible biological or ecophysiological roles of toxin production. Toxins might
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The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (ED) and its 27-nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.
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The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC50 for hemolysis of a sonicated cell suspension was 2.4×104 cells ml−1, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC50 for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml−1 while the LC50 of the second, smaller, fraction (Tox B) was 600 ng ml−1. For comparison, the LC50 for the standard hemolysin saponin was 3203 ng ml−1. At concentrations of 800 and 2000 ng ml−1, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml−1 Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml−1. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.