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Generation of Reactive Oxygen Species (ROS) by Harmful Algal Bloom (HAB)-Forming Phytoplankton and Their Potential Impact on Surrounding Living Organisms

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Most marine phytoplankton with relatively high ROS generation rates are categorized as harmful algal bloom (HAB)-forming species, among which Chattonella genera is the highest ROS-producing phytoplankton. In this review, we examined marine microalgae with ROS-producing activities, with focus on Chattonella genera. Several studies suggest that Chattonella produces superoxide via the activities of an enzyme similar to NADPH oxidase located on glycocalyx, a cell surface structure, while hydrogen peroxide is generated inside the cell by different pathways. Additionally, hydroxyl radical has been detected in Chattonella cell suspension. By the physical stimulation, such as passing through between the gill lamellas of fish, the glycocalyx is easily discharged from the flagellate cells and attached on the gill surface, where ROS are continuously produced, which might cause gill tissue damage and fish death. Comparative studies using several strains of Chattonella showed that ROS production rate and ichthyotoxicity of Chattonella is well correlated. Furthermore, significant levels of ROS have been reported in other raphidophytes and dinoflagellates, such as Cochlodinium polykrikoides and Karenia mikimotoi. Chattonella is the most extensively studied phytoplankton in terms of ROS production and its biological functions. Therefore, this review examined the potential ecophysiological roles of extracellular ROS production by marine microalgae in aquatic environment.
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Citation: Cho, K.; Ueno, M.; Liang, Y.;
Kim, D.; Oda, T. Generation of
Reactive Oxygen Species (ROS) by
Harmful Algal Bloom
(HAB)-Forming Phytoplankton and
Their Potential Impact on
Surrounding Living Organisms.
Antioxidants 2022,11, 206.
https://doi.org/10.3390/
antiox11020206
Academic Editor: Stanley Omaye
Received: 29 December 2021
Accepted: 20 January 2022
Published: 22 January 2022
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antioxidants
Review
Generation of Reactive Oxygen Species (ROS) by Harmful
Algal Bloom (HAB)-Forming Phytoplankton and Their
Potential Impact on Surrounding Living Organisms
Kichul Cho 1, Mikinori Ueno 2, Yan Liang 2, Daekyung Kim 3,* and Tatsuya Oda 2, *
1Department of Microbiology, National Marine Biodiversity Institute of Korea (MABIK),
Seocheon 33662, Korea; kichul.cho@mabik.re.kr
2Graduate School of Fisheries Science & Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan; mikiueno@nagasaki-u.ac.jp (M.U.); bb53419804@ms.nagasaki-u.ac.jp (Y.L.)
3Daegu Center, Korea Basic Science Institute (KBSI), Daegu 41566, Korea
*Correspondence: dkim@kbsi.re.kr (D.K.); t-oda@nagasaki-u.ac.jp (T.O.)
Abstract:
Most marine phytoplankton with relatively high ROS generation rates are categorized as
harmful algal bloom (HAB)-forming species, among which Chattonella genera is the highest ROS-
producing phytoplankton. In this review, we examined marine microalgae with ROS-producing
activities, with focus on Chattonella genera. Several studies suggest that Chattonella produces superox-
ide via the activities of an enzyme similar to NADPH oxidase located on glycocalyx, a cell surface
structure, while hydrogen peroxide is generated inside the cell by different pathways. Additionally,
hydroxyl radical has been detected in Chattonella cell suspension. By the physical stimulation, such as
passing through between the gill lamellas of fish, the glycocalyx is easily discharged from the flagel-
late cells and attached on the gill surface, where ROS are continuously produced, which might cause
gill tissue damage and fish death. Comparative studies using several strains of Chattonella showed
that ROS production rate and ichthyotoxicity of Chattonella is well correlated. Furthermore, significant
levels of ROS have been reported in other raphidophytes and dinoflagellates, such as Cochlodinium
polykrikoides and Karenia mikimotoi.Chattonella is the most extensively studied phytoplankton in
terms of ROS production and its biological functions. Therefore, this review examined the potential
ecophysiological roles of extracellular ROS production by marine microalgae in aquatic environment.
Keywords:
reactive oxygen species (ROS); marine microalgae; harmful algae bloom (HAB) species;
Chattonella; nitric oxide (NO)
1. Introduction
Over the years, hundreds of marine microalgae, which constitute a significant por-
tion of marine biomass, have been described worldwide. Although several microalgae
are known as promising sources of beneficial bioactive compounds, such as anti-cancer
and antioxidant agents [
1
,
2
], less than 2% of them have been classified as harmful or
toxic species [
3
]. The blooming of dominant algae species at relatively high cell density
results in a phenomenon called “red tides”. In the case of harmful or toxic species, it is
known as harmful algal blooms (HABs) [
4
]. The mass growth of specific algal species is
easily recognized, often leading to water discoloration [
4
]. Most HAB species in marine
environments are unicellular phytoplankton. HAB is a diverse phenomenon, involving
multiple species and classes of microalgae that produce a wide variety of toxins or bioac-
tive compounds, which can negatively affect several aquatic organisms [
4
6
]. Owing to
advances in monitoring, surveillance, and identification technology, there has been an
increase in the number of identified harmful and toxic microalgae species. Moreover, there
has been an increased awareness of HABs due to an increase in the utilization of coastal
areas for aquacultural activities [
7
,
8
]. Globalization of logistics seems to contribute to the
Antioxidants 2022,11, 206. https://doi.org/10.3390/antiox11020206 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 206 2 of 28
distribution of HAB species to new areas through ballast water of ships or contaminated
seafoods [
9
12
]. Recently, there has been an increase in the impact of HABs on fisheries
and aquaculture industries. Moreover, the influence of HABs on marine ecosystems and
organisms is projected to increase under the present climate change scenario, which could
lead to considerable economic and ecological implications.
HABs have considerable implications on commercial and recreational fisheries and
coastal tourism, as well as human and wildlife health [3,1317].
There is emerging evidence that global warming-induced environmental changes may
influence the patterns, frequency, distribution, and intensity of HABs in marine, brackish,
and freshwater environments [
18
21
]. Changes in temperature [
13
], ocean acidification [
22
],
nutrient conditions [
23
], and the physical structure of the water column [
24
], have been
shown to influence the incidence and global range of HABs.
One of the significant impacts of HABs is the accumulation of algal toxins in shellfish,
resulting in food poisoning of humans and animals. Symptoms of shellfish poison in
humans include paralytic, diarrhetic, neurotoxic, amnesic, and azaspiracid poisoning [
25
].
Additionally, toxins and other bioactive compounds produced by algal species during
bloom can cause the death of marine organisms. Furthermore, HABs can cause the death
of wildlife, including seabirds, whales, dolphins, and other marine animals, through the
transfer of toxins via the food web or direct ingestion of toxins [
3
,
16
]. Major incidences of
HABs-associated mass mortalities and huge economic losses are summarized in
Table 1
.
Although most of the toxins responsible for shellfish poisoning are well defined, the
mechanisms of lethal action of harmful species are poorly understood. Overall, raphido-
phytes and dinoflagellates are the main groups of HAB species. Particularly, species of
the Chattonella genera (C. marina,C. antiqua,C. subsalsa,C. minima, and C. ovata) have been
reported to cause severe mortality of farmed organisms in mainly temperate and adjacent
waters [2632]
. Yellowtail (Seriola quinqueradiata), atlantic salmon (Salmo salar), northern
bluefin tuna (Thunnus orientalis), and bluefin tuna (Thunnus maccoyii) are especially suscep-
tible to
Chattonella [3237]
. Mortalities have also been reported in benthic organisms, such
as blue crabs, clams, octopus, pen shells, shrimps, and sea cucumber during Chattonella
blooms [
38
]. In Japan, Chattonella have been reported to cause economic loss of approxi-
mately JPY 7.1 billion in the Harima-nada marine region in 1972 [
39
], and JPY 2.9 billion in
2009 and JPY 5.3 billion in 2010 in the Yatsushiro Sea [
40
]. Additionally, economic losses to
fisheries due to Chattonella have been reported in other countries [
25
,
30
32
,
35
,
37
,
38
,
41
47
].
Table 1.
Harmful effect of HAB-forming species and their toxins (directly citing reviews of
Landsberg [3]).
Species Main Toxic Factors Event References
Karenia mikimotoi Hemolysin; Reactive oxygen
species (ROS)
Ichthyotoxic; Toxic to invertebrates;
Toxic to zooplankton; Antialgal [4856]
Cochlodinium polykrikoides Hemolysin; ROS; Sulfated
polysaccharides; Noxiustoxin
Antiviral; Ichthyotoxic;
Molluscicidal [5762]
Eutreptiella gymnastica ROS - [62]
Alexandrium tamarense
Saxitoxin; Neosaxitoxin;
Gonyautoxin; N-sulfocarbomoyl
toxins; Tetrodotoxin;
Hemolysin; ROS
Neurotoxic; Paralytic shellfish
poisoning; Toxic to marine
organisms; Toxic to zooplankton;
Cytotoxic
[6383]
Heterosigma akashiwo
(=Heterosigma carterae) Brevetoxin-like toxin; ROS Ichthyotoxic; Neurotoxic; Toxic to
zooplankton [8489]
Chattonella marina Brevetoxin-like toxin
ROS; Hemolysin; Hemagglutinin Ichthyotoxic; Neurotoxic [9,88,9094]
Chattonella ovata ROS Ichthyotoxic [95]
Chattonella antiqua Brevetoxin-like toxin; ROS Ichthyotoxic; Neurotoxic [96100]
Chattonella subsalsa ROS; Hemolysin Ichthyotoxic; Toxic to zooplankton [101]
Platymonas subcordiformis ROS - [102]
Antioxidants 2022,11, 206 3 of 28
Table 1. Cont.
Species Main Toxic Factors Event References
Skeletonema costatum ROS Ichthyotoxic; Toxic to zooplankton;
Antibacterial [103106]
Olisthodiscus luteus ROS Ichthyotoxic; Antimycotic; Toxic to
phytoplankton [88,98,107,108]
Fibrocapsa japonica Brevetoxin-like toxin; ROS;
Fibrocapsin
Ichthyotoxic; Toxic to marine
mammals;
Neurotoxic; Toxic to phytoplankton
[88,107,109]
Heterocapsa circularisquama ROS; Hemolysin Toxic to mollusks; Antialgal;
Antiprotozoal; Toxic to zooplankton
[110115]
Akashiwo sanguineum
(=Gymnodinium sanguineum) ROS Ichthyotoxic; Toxic to mollusks;
Antimycotic; Toxic to mice [55,98,116,117]
Karlodinium veneficum ROS Ichthyotoxic; Toxic to zooplankton [55,118,119]
Alexandrium catenella
Saxitoxin; Neosaxitoxin;
Gonyautoxin; N-sulfocarbomoyl
toxins; Hemolysin; ROS
Neurotoxic; Paralytic shellfish
poisoning; Toxic to marine
organisms
[73,82,120126]
Prorocentrum minimum Venerupin; Prorocentrin;
ß-diketone; ROS
Venerupin shellfish poisoning;
Toxic to marine organism;
Neurotoxic
[127139]
Prymnesium parvum Prymnesin 1 and 2; Hemolysin
Ichthyotoxic; Toxic to invertebrates;
Toxic to tadpoles; Toxic to
zooplankton; Cytotoxic
[140145]
Thalassiosira weissflogii ROS Toxic to zooplankton [105]
Thalassiosira pseudonana
Apo-fucoxanthinoid pigments; ROS
Toxic to zooplankton [146,147]
Coscinodiscus sp. ROS - [148]
Pleurochrysis carterae ROS Toxic to zooplankton [149]
Symbiodinium spp. ROS - [150]
Trichodesmium erythraeum ROS
Antibacterial; Toxic to marine
organisms; Hepatotoxic;
Neurotoxic; Ciguatoxin-like
[151154]
Prorocentrum micans ROS
Shellfish poisoning; Toxic to marine
organisms; Antialgal [62,155158]
Although several studies have been performed on algal bloom, the exact toxic mech-
anisms of harmful algae are yet to be elucidated. Previous studies on the ichthyotoxic
mechanism of Chattonella have identified several toxic or bioactive compounds involved in
fish mortality [
39
,
100
,
159
165
] or the synergistic impact of multiple toxic factors [
166
]. It has
been widely accepted that suffocation is the major cause of fish death by
Chattonella [167170]
.
Previous findings suggest that the direct target organ of Chattonella is the gill tissue, which
can eventually lead to fish death. Matsusato and Kobayashi reported that the dead cells
of C. antiqua and cell-free filtrate prepared from the live cell suspension of Chattonella was
non-toxic to fish (red sea bream) [
171
]. Similarly, Ishimatsu et al. [
172
] also found that lysed
cells of C. marina did not kill yellowtail.
Other raphidophytes, such as Heterosigma akashiwo,Olisthodiscus luteus, and Fibrocapsa
japonica, also produce extracellular ROS [
85
,
88
,
160
,
173
,
174
]. Thus, it seems that ROS pro-
duction is a common biological feature of raphidophytes. However, extracellular ROS
production is not limited to raphidophytes and has been reported in harmful dinoflagel-
lates, including Alexandrium spp. [
173
,
175
177
], Margalefidinium polykrikoides (Cochlodinium
polykrikoides) [62,178180], and Karenia mikimotoi [176,181183].
A recent review showed that phytoplankton are a major producer of ROS, including
superoxide and hydrogen peroxide in aquatic environments [
184
]. Many phytoplankton
taxa generate ROS under ordinal growth conditions without any stimuli or stress conditions.
Although the physiological significance of extracellular ROS production by phytoplankton
and their effects on the ecosystem remain unclear, the potential ecological and physiological
effects of ROS production include biotoxicity, allelopathy, growth promotion, and iron
acquisition. In this review, we described the levels, subcellular mechanism, biological roles,
Antioxidants 2022,11, 206 4 of 28
and toxic potential of ROS production by HABs species, with emphasis on the Chattonella
genera [
184
]. Different assay methods have been applied for the detection of ROS produced
by marine microalgae, as shown in Table 2.
Additionally, several lines of evidence suggest that C. marina can produce nitric
oxide (NO) [
185
], which is involved in various important biological processes in mam-
mals [
186
], several metabolisms of plants, expression of gene [
187
], and infectious diseases
in plants [
188
,
189
]. Moreover, NO and superoxide can form peroxynitrite, a potent oxidant,
by reacting to each other. Thus, the mechanism of NO production by Chattonella and other
species and their biological activities was discussed in the later part of this review.
Table 2.
List of assay methods used for the detection of reactive oxygen species (ROS) in microalgae.
ROS Studied Algal Species Methods References
Superoxide
(O2)
Chattonella marina
Chattonella antiqua
Karenia mikimotoi
Cochlodinium polykrikoides
Chattonella ovata
Olisthodiscus luteus
1MCLA-mediated chemiluminescence assay [62,94,159,160,166,181,
190192]
Chattonella marina
Chattonella antiqua
Karenia mikimotoi
Cochlodinium polykrikoides
Chattonella ovata
2L012-mediated chemiluminescence assay [164,193195]
Cochlodinium polykrikoides
Heterosigma carterae
Thalassiosira weissflogii
Thalassiosira pseudonana
Cytochrome c-mediated
spectrophotometric assay [62,85,196]
Chattonella marina
Nostoc spongiaeforme
3DMPO-mediated spin trapping method
using an ESR [159,197,198]
Trichodesmium erythraeum 4Red-CLA-mediated chemiluminescence [199]
Hydroxyl radical
(OH)Chattonella marina Phenol red assay [161]
Hydrogen peroxide
(H2O2)
Chattonella marina
Cochlodinium polykrikoides
3DMPO-mediated spin trapping method
using an ESR [159,198]
Karenia mikimotoi
Cochlodinium polykrikoides
Chattonella ovata
5PHPA-mediated fluorescence
spectrophotometric assay [178,181,200]
Cochlodinium polykrikoides Scopoletin–peroxidase method [62,201]
Nitric oxide
Chattonella marina Cochlodinium
polykrikoides Phenol red assay [178,202]
Chattonella marina Luminol–H2O2-mediated luminescence assay [185,203]
Chattonella marina
Heterosigma akashiwo
Chatonella ovata
Cochlodinium polykrikoides
Alexandrium taylori
Alexandrium tamarense
Nannochloropsis oculata
6DAF-FM DA-mediated fluorometric assay [204]
Nitric oxide
Platymonas subcordiformis
Skeletonema costatum
Gymnodinium sp.
Nitric oxide detection microsensor [102]
1
MCLA, methyl cypridina luciferin analog;
2
L012, 8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-
1,4-(2H,3H)-dione;
3
DMPO, 5,5-dimethyl-1-pyrroline N-oxide;
4
red CLA, [2-[4-[4-[3,7-dihydro-2-methyl-3-
oxoimidazo[1,2-a]pyrazin-6-yl]phenoxy]butyramido]ethylamino]sulforhodamine 101;
5
PHPA, p-hydroxyphenyl
acetic acid; 6DAF-FM DA, 4-Amino-5-methylamino-20,70-difluorofluorescein diacetate.
2. Marine Microalgae Species with ROS-Producing Activities
In 1989, Shimada et al. [
205
] reported the first evidence of ROS production by the raphi-
dophycean flagellate Chattonella antiqua. Since then, numerous studies have been conducted
Antioxidants 2022,11, 206 5 of 28
on Chattonella spp., including the role of ROS as an ichthyotoxic factor, ROS production
mechanism, and the biological roles of ROS in Chattonella [
28
,
31
,
206
,
207
]. Despite extensive
studies [
86
,
88
,
93
,
100
,
159
,
160
,
166
,
171
,
208
212
], the exact mechanism through which Chat-
tonella cause fish death is still poorly understood. Recently, Shikata et al. [
213
] examined the
ichthyotoxicity of eight strains of Chattonella with different backgrounds against different
fish species (red sea bream and yellowtail) and found that the generation level of superoxide
was most well-correlated with fish-killing activity among several factors examined, which
supports the notion that ROS are mainly involved in the Chattonella-related fish mortality.
Moreover, few studies have found that other raphidophytes [
85
,
88
,
173
,
174
] and some
dinoflagellates [
62
,
173
,
175
183
] are capable of producing ROS. Furthermore, Marshall
et al. [
173
] examined the superoxide-producing ability of 37 species of microalgae, such
as dinoflagellates, raphidophytes, and others, using chemiluminescence analysis, and
found that several phytoplankton species are capable of producing superoxide to some
extent. Detailed analyses showed a direct correlation between cell size and superoxide
production level. Among the species, Chattonella produced the highest levels of superoxide
per cell, whereas harmless species, such as Dunaliella,Tetraselmis,Nannochloropsis, and
Pavlova, which are usually used as bivalve feeds, did not produce significant levels of ROS.
Furthermore, based on the degree of superoxide production and toxicity, they proposed
that phytoplankton species could be classified into four groups. Microalgae producing ROS
with exceled certain threshold value, such as C. antiqua,C. marina,C. minima and C. ovata,
were categorized as extremely toxic.
According to a review paper on the diversity of phytoplankton in aquatic environ-
ments [
184
], the generation rate of ROS per cell was measured in more than 21 microalgae
species; most of them were HAB-forming species [
62
,
85
,
173
,
175
,
182
]. The generation rate
of one ROS (superoxide; O
2
) has also been quantified in some species of cyanobacte-
ria [
148
,
199
,
214
216
]. Furthermore, HAB species produce higher levels of O
2
than other
phytoplankton taxa, including freshwater cyanobacterium Microcystis aeruginosa [
216
], and
non-harmful species [
196
]. These findings indicate that various phytoplankton species
are major biological sources of ROS in marine environment, which can cause profound
ecological impact on marine environment. Considering the high level of ROS production
in marine environments, it is necessary to examine the effects of ROS produced by HAB
species, including raphidophytes and dinoflagellates, on marine organisms. Therefore,
Chattonella spp., other raphidophytes, and dinoflagellates, such as Cochlodinium polykrikoides
and Karenia mikimotoi, were discussed comprehensively in subsequent sections. Table 3
shows details of high ROS-producing marine phytoplankton species.
Table 3.
Reactive oxygen species (ROS)-producing microalgae and the estimated production
mechanisms.
Algal Species ROS Estimated Production Mechanisms References
Karenia mikimotoi Superoxide
Hydrogen peroxide - [181,182]
Cochlodinium polykrikoides
Superoxide
Hydrogen peroxide
Hydroxyl radical
Auto-oxidation of an electron
acceptor in photosystem I (superoxide)
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
[159,178,198,217]
Eutreptiella gymnastica Hydrogen peroxide SOD catalyzed disproportionation of
superoxide (hydrogen peroxide) [62,184]
Prorocentrum micans Hydrogen peroxide SOD catalyzed disproportionation of
superoxide (hydrogen peroxide) [62]
Akashiwo sanguineum
(=Gymnodinium sanguineum) Hydrogen peroxide SOD catalyzed disproportionation of
superoxide (hydrogen peroxide) [62]
Alexandrium tamarense Hydrogen peroxide SOD catalyzed disproportionation of
superoxide (hydrogen peroxide) [62]
Antioxidants 2022,11, 206 6 of 28
Table 3. Cont.
Algal Species ROS Estimated Production Mechanisms References
Heterosigma akashiwo
(=Heterosigma carterae)
Superoxide
Hydrogen peroxide
Hydroxyl radical
Nitric oxide
Glycocalyx-mediated ROS generation
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
[62,85,162,218]
Chattonella marina
Superoxide
Hydrogen peroxide
Nitric oxide
NAD(P)H oxidase located in cell
surface-bounded glycocalyx (superoxide)
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
Nitric oxide synthase-like
enzyme-mediated mechanism (nitric
oxide)
[94,162,185,203]
Chattonella ovata
Superoxide
Hydrogen peroxide
Nitric oxide
NAD(P)H oxidase located in cell
surface-bounded glycocalyx (superoxide)
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
[162]
Chattonella antiqua Superoxide
Hydrogen peroxide
NAD(P)H oxidase located in cell
surface-bounded glycocalyx (superoxide)
Photosynthetic electron transport
(superoxide)
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
[193,219]
Chattonella subsalsa Superoxide Photosynthetic electron transport
(superoxide) [217]
Platymonas subcordiformis Superoxide - [102]
Skeletonema costatum Superoxide - [102]
Olisthodiscus luteus Superoxide
Hydrogen peroxide
Cell surface redox enzyme-mediated
mechanism (superoxide)
SOD catalyzed disproportionation of
superoxide (hydrogen peroxide)
[108]
Fibrocapsa japonica Superoxide
Hydrogen peroxide - [174]
Heterocapsa circularisquama
Karlodinium veneficum
Hydrogen peroxide
Superoxide
-[182,184]
-
Alexandrium catenella Superoxide - [184]
Prorocentrum minimum Hydrogen peroxide - [184]
Prymnesium parvum Superoxide - [184]
Thalassiosira weissflogii Superoxide
Hydrogen peroxide
NAD(P)H oxidase-related mechanism
[196,220]
Thalassiosira pseudonana Superoxide
Hydrogen peroxide
NAD(P)H oxidase-related mechanism
[196]
Thalassiosira oceanica Superoxide
Hydrogen peroxide
NAD(P)H oxidase-related mechanism
[221]
Coscinodiscus sp. Superoxide - [184]
Pleurochrysis carterae Hydrogen peroxide - [222]
Symbiodinium spp. Superoxide - [150,223]
Trichodesmium erythraeum Superoxide - [199]
3. Chattonella
Raphidophycean flagellates Chattonella spp (C. marina,C. antiqua,C. subsalsa,C. minima,
and C. ovata) are causative species of HAB-associated fish mortality, with serious impact
on the aquacultural industry in Japan [
31
]. Among the genus, C. marina and C. antiqua are
highly toxic species, which are causing enormous negative impact on fish farms in Japan,
particularly to yellowtail (Seriola quinqueradiata) aquaculture in the last few decades [
212
].
Additionally, fish mortality due to Chattonella spp-induced HABs has occurred in Australia,
Netherlands, Brazil, and other parts of the world [28,31,206,207].
Previous studies have proposed several potential toxic factors, such as neurotoxins re-
sembling the brevetoxins produced by Karenia brevis (formerly known as Gymnodinium breve
Antioxidants 2022,11, 206 7 of 28
and Ptychodiscus brevis), haemagglutinating agents [
86
,
93
,
100
,
210
,
211
], fatty acids [
166
,
212
],
and mucus substances [
171
]. Moreover, Shimada et al. [
163
] reported that C. antiqua has the
ability to induce SOD-inhibitable cytochrome c reduction, indicating that live C. antiqua
cells can produce superoxide anion. Further studies using several techniques demon-
strated that Chattonella spp. generate ROS, such as superoxide (O
2
), hydrogen peroxide
(H
2
O
2
), and hydroxyl radical (
OH) [
88
,
159
,
160
,
208
,
209
]. Since ROS are biologically highly
toxic [
224
,
225
], Chattonella spp. may exert ichthyotoxicity through ROS at least in part. This
hypothesis might be supported by the low toxicity of low superoxide-producing C. marina
strains [
172
,
226
]. Similarly, Cho et al. [
164
] reported relatively high ichthyotoxicity of high
ROS-producing C. antiqua strains compared with low ROS strain of C. marina. In addition
to Chattonella spp., another raphidophycean flagellate, Heterosigma akashiwo, has been re-
ported to show ROS-mediated toxicity against rainbow trout [
85
]. It is widely accepted that
suffocation is the main mechanism of fish death by these flagellates [
167
170
], with loss
of branchial respiratory capacity as an immediate physiological change observed in fish
after exposure to Chattonella spp. [
31
,
227
]. Exposure of S.quinqueradiata to C. marina at a
lethal cell density causes a rapid decrease in the arterial oxygen pressure within less than
30 min [
168
,
169
,
228
], resulting in further physiological responses, such as acidosis [
229
],
ionoregulatory failure [
168
], increase in circulating catecholamine levels [
230
], and decrease
in cardiac output [
228
]. Owing to a decrease in arterial oxygen pressure, excess mucus-like
substances are secreted, probably by gill tissues, in response to stimulus by C. marina,
which cover the gills. Such mucus substance on the gill surface together with glycocalyx, a
polysaccharide-containing complex cell surface structure discharged from the flagellate
cells, may interfere with O
2
uptake from gill lamellas, resulting in asphyxia [
169
,
231
,
232
].
A further
in vitro
study demonstrated that there was a 26–83% decrease in water flow rate
through the excised first gill arch of jack mackerel (Trachurus japonicus) placed in 4000 cells
of C. marina/mL for 10 min compared with those placed in culture medium alone, and the
gill arch of the flagellate-exposed group was covered with mucus and C. marina cells [
233
].
In mammalian systems, ROS enhance mucus secretion from various epithelia lining luminal
organs, such as the gallbladder of guinea pigs
in vitro
[
234
,
235
], the gastric mucous cells
of rats [
236
], and the tracheal epithelial cells of guinea pigs [
237
,
238
]. Considering the
similarities between fish and mammals in terms of mucins, mucus cells [
239
], and secretory
mechanisms [
240
], it could be speculated that ROS produced by C. marina may be involved
in the over secretion of mucus on the gill tissue.
Based on previous findings, it seems likely that live cell condition is important for the
ichthyotoxicity of Chattonella. Matsusato and Kobayashi [
171
] reported that neither the
dead cells of C. antiqua nor cell-free supernatant of the flagellate culture were toxic to fish.
Similarly, Ishimatsu et al. [
172
] reported that ruptured C. marina showed no toxic effect on
yellowtail and found that there is a clear correlation between the cellular O
2
producing
activity and its fish toxicity. The toxin may probably be quite unstable in nature, leading to
the disappearance of its activity in ruptured cells and rendering the isolation of the toxin in
its active form difficult. Considering these findings, among the toxic factors proposed, ROS
is the most provable candidate.
Previous studies demonstrated that C. marina suppressed the growth of Vibrio algi-
nolyticus inoculated into plankton culture [
160
]. The bactericidal activity of C. marina was
significantly suppressed by superoxide dismutase (SOD) and catalase, which are antioxi-
dant enzymes with ROS scavenging activity. Additionally, sodium benzoate, a hydroxyl
radical scavenger, protected the bacteria from the toxic effect of C. marina. Thus, it was
suggested that C. marina can exert negative impact on surrounding bacteria through ROS
production. This is an indicative example that ROS-producing marine microalgae, such as
Chattonella spp., can cause oxidative stress to surrounding organisms.
3.1. Mechanisms of ROS Production by Chattonella
Various phytoplankton species produce extracellular ROS under normal growth con-
ditions, with Chattonella marina being the highest extracellular ROS producer [
184
]. ROS pro-
Antioxidants 2022,11, 206 8 of 28
duction by Chattonella has been well documented in several independent
studies [159161,163,164,226]
. Several techniques have been employed for the detection of
each reactive oxygen species; superoxide anions (O
2
) by cytochrome c reduction [
160
],
chemiluminescence analysis [
164
], and fluorescent microscopy [
162
,
163
]; hydrogen perox-
ide (H
2
O
2
) by the phenol red or the scopoletin assay [
161
]; and superoxide and hydroxyl
radicals (
OH) by electron spin resonance spectroscopy [
159
,
164
]. In Chattonella cells, ROS
production can occur in several major organelles or intracellular compartments, such as
chloroplasts, mitochondria, peroxisomes, and cell membrane. The primary oxygen radical-
producing step at these sites is the formation of O
2
via the single electron reduction of
O
2
, and the subsequent enzymatic or non-enzymatic dismutation of superoxide is the
most probable mechanism for the production of H
2
O
2
. Intracellularly generated H
2
O
2
might easily release extracellularly [
94
,
162
]. In contrast, O
2
is membrane impermeable
in nature due to its short life span and limited diffusion distance, indicating that O
2
hardly crosses cell membranes [
241
,
242
]. Thus, the most probable site of O
2
generation
in Chattonella may be on the cell surface. To identify the mechanism of O
2
and H
2
O
2
production, especially focusing on intracellular location of O
2
and H
2
O
2
production in
C. marina and C. ovata, Kim et al. [162] conducted fluorescence microscopic observation of
these flagellate cells using methyl cypridina luciferin analog and 5-(and-6)-carboxy-20,70-
dichlorodihydrodihydrofluorescein dictate, acetyl ester, which is a specific fluorescent
probe for detecting O
2
and H
2
O
2
, respectively. The fluorescence pictures suggested
that superoxide is produced on the cell surface, whereas hydrogen peroxide is produced
intracellularly. Furthermore, destruction of the cells by ultrasonic treatment resulted in
significant decrease in O
2
levels, whereas the level of H
2
O
2
detected in the ruptured cells
increased as compared to the level of intact cell suspension [
161
,
162
]. Thus, the producing
mechanisms of O
2
and H
2
O
2
and their intracellular location seem to be different and
independent of each other in the cells.
Generally, it has been considered that hydroxyl radical is the most toxic radical that can
destroy proteins, nucleic acids, and other important biomolecules [
224
,
243
]. Since hydroxyl
radical is detected in the flagellate cells, the ecological impact of these ROS-producing
flagellates should be significant. The reaction of superoxide radical and hydrogen peroxide
can produce hydroxyl radical. For this reaction, transition metals, such as Fe
2+
and Cu
2+
,
play an important role as reducing agents in the Fenton reaction and the Haber–Weiss
cycle [
224
,
243
]. Iron is generally required for optimal growth of phytoplankton [
244
],
and the flagellate culture medium contains 0.5
µ
M EDTA-Fe
3+
and certain levels of other
metal ions. Thus, it is possible that the hydroxyl radical is produced through the Fe-
catalyzed Fenton-type Haber–Weiss reaction. This assumption was supported by the
fact that hydroxyl radical production in flagellate cell suspension is inhibited by either
SOD or catalase [
159
]. To further evaluate the roles of iron or other metals in hydroxyl
radical production, the effect of hypoxanthine/xanthine oxidase addition as a superoxide
generation system to flagellate culture medium was examined using electron spin resonance
(ESR) analysis, and the results showed that hypoxanthine/xanthine oxidase remarkably
increased hydroxyl radical production [
159
]. Because Fe exists in seawater in the 0.01–1
µ
M
range [245], the formation of hydroxyl radical by Chattonella spp. in seawater is feasible.
Tang et al. [
246
] showed that H
2
O
2
alone was not lethal to fish. It should be noted
that Chattonella cells produce both superoxide anions and hydrogen peroxide, and the
co-occurrence enhances Chattonella toxicity to living organisms, which could be due to
hydroxyl radical formation [
247
,
248
]. Additionally, studies have shown that H
2
O
2
had
no effect on mucin release in several cell culture or explant models [
235
,
237
,
249
], whereas
hydroxyl radical induced mucus secretion [
235
]. Since the presence of excessive mucus
substance on the gill surface of fish exposed to Chattonella is considered as a key factor, hy-
droxyl radical may play a major role among the ROS in terms of ROS-mediated detrimental
effect of Chattonella on fish gill.
Antioxidants 2022,11, 206 9 of 28
3.2. NADPH Oxidase as a Superoxide-Anion-Producing Enzyme System
In various biological systems, the generation of extracellular superoxide anion (O
2
)
is likely regulated by enzyme systems, such as oxidoreductases, utilizing nicotinamide
adenine dinucleotide phosphate (NADPH), which acts as a reducing co-factor for the
conversion of O
2
to O
2
. An enzyme NADPH oxidase existing in the plasma membrane
of certain white blood cells catalyzes the single-electron reduction of O
2
to O
2
[
250
]. In
higher plant cells, there are some NAD(P)H oxidase capable of generating O
2
in the
plasma membranes, and ROS production in plant cells shows similar characteristic of
ROS-generation system so-called oxidative burst in mammalian phagocytic cells [251].
Raphidophytes (C. antiqua,C. marina,H. akashiwo) possess glycocalyx as a cell surface
structure [
232
,
252
254
], and enzymatic system responsible for O
2
generation exists in the
glycocalyx, which is easily dissociated from the cells under physical or chemical stimula-
tion [
97
,
191
]. To determine the involvement of glycocalyx in ROS generation in C. marina,
Kim et al. obtained a supernatant from a C. marina cell suspension by mild agitation, which
caused discharge of the glycocalyx without cell destruction [
94
]. Chemiluminescence assay
using O
2
specific probe showed that the cell-free supernatant induced SOD-inhibitable
strong chemiluminescence in response to exogenous NADPH, whereas the supernatant
without NADPH showed only a trace-level response. Additionally, concentration of high
molecular weight fraction by ultrafiltration resulted in increased chemiluminescence re-
sponse, suggesting that certain components with large molecular size in the supernatant
are responsible for the reaction. On the other hand, C. marina cell suspension exhibited no
response to NADPH. Since NADPH is not membrane permeable, C. marina might not be
able to utilize extracellular NADPH. NADH was less effective as compared to NADPH,
and NADP
+
and NAD
+
were ineffective. In addition, diphenyleneiodonium, an inhibitor
of mammalian NADPH oxidase, prevented NADPH-induced chemiluminescence response
in the cell-free supernatant. Probably, C. marina has an enzyme system similar to NADPH
oxidase of neutrophil. NADPH oxidase of neutrophil has two subunit proteins in the
plasma membrane, gp91phox and p22phox, which form heterodimeric flavocytochrome
b558 [
250
]. To further clarify the O
2
-generating enzyme system in C. marina cells, im-
munoblotting of the cell-free supernatant of C. marina was performed using an antibody
raised against neutrophil gp91phox. The result suggested the presence of protein recog-
nized with the antibody in the cell-free supernatant of C. marina. Additionally, indirect
immunofluorescence of the flagellate cells using the same antibody indicated that human
gp91phox-like protein existed on the surface of C. marina. Furthermore, southern blot using
the oligonucleotide probe encoding the C-terminal region of human gp91phox suggested
the presence of a gene encoding a protein mimicking gp91phox in C. marina. In addition
to several well-described mammalian homologs of gp91phox [
255
257
], higher plant cells
(Arabidopsis thaliana) have slightly larger homologs of gp91phox with 59.8–62.3% sequence
similarity to gp91phox [258].
The presence of NADPH oxidase in Chattonella as a source of O
2
is further supported
by the identification of six putative genes encoding NADPH oxidase (NOX) in C. anti-
qua [
259
]. The enzymatic activity of NOX requires NADPH, which is mainly supplied
by the oxidative pentose phosphate (OPP) pathway [
260
,
261
]. Regarding the regulation
mechanism of NOX activity in Chattonella, it has been observed that the production of O
2
in C. marina and C. antiqua is inhibited by an inhibitor of photosynthetic electron transport,
3-(3,4-dichlorophenyl)-1,1-dimethylurea [
193
,
262
]. These findings suggest that both pho-
tosynthesis and OPP pathways are involved in the production of O
2
in these flagellate
cells. Interestingly, a recent study found that O
2
production in C. antiqua increased under
nutrient deficiency and suppressed photosynthesis conditions, suggesting that increases in
the ratio of NADPH to NADP
+
caused by the OPP pathway might be deeply involved in
ROS generation in Chattonella [263].
Antioxidants 2022,11, 206 10 of 28
3.3. Glycocalyx as a Cell Surface Structure with ROS Generation System
Electron and light microscopic observation of Chattonella antiqua and Heterosigma
akashiwo showed that these cells have glycocalyx as the cell surface, which consists of sul-
fated, non-sulfated polysaccharides, and neutral carbohydrate–protein
complex [252,253]
.
Since raphidophycean flagellates generally do not have a rigid cell wall, glycocalyx may
function as a defense or barrier against biological and non-biological invasion.
Oda et al. [191]
showed that the addition of lectins, such as concanavalin A (Con A), wheat germ agglutinin,
and castor bean haemagglutinin, significantly increased in O
2
generation by C. marina
and H. akashiwo. Since the effects of the lectins were suppressed by specific monosaccha-
rides, the binding of the lectins to the saccharide moieties on the cell surface may have
led to increased O
2
production. Interestingly, high concentration of Con A can induce
morphological changes in these flagellate cells. After the addition of Con A, some cells
became spherical, distinct from the usual spindle shape, and these changes were frequently
accompanied by the discharge of glycocalyx. An analysis using fluorescent-labeled Con A
confirmed the binding of Con A to the discharged glycocalyx. These results suggest that
the binding of Con A to the glycocalyx is recognized as a stimulus by the flagellate cells,
leading to discharge of glycocalyx. Shimada et al. [
163
] and Tanaka et al. [
99
] reported
that O
2
was generated in small particles, or in verruciform protrusions located on the
cell surface of C. antiqua. Additionally, the addition of mucus substances prepared from
yellowtail induced the release of these small particles from the flagellate cells. Similarly,
Nakamura et al. [
218
] and Okamoto et al. [
254
] observed that extracellular addition of mu-
cus substances obtained from yellowtail gill enhanced O
2
generation by C. marina, which
was concomitant with the discharge of the glycocalyx. The presence of O
2
generation
system on the glycocalyx, may be supported by the observed suppression of O
2
gener-
ation by C. marina and H. akashiwo treated with membrane-impermeable protease [
191
].
In fish mucus, lysozyme, proteases, and lectins and other bioactive molecules have been
discovered, as well as mucin, a major mucus comportment [
264
266
]. Some components in
fish mucus that possess lectin activity may act as a stimulus mimicking Con A and induce
glycocalyx discharge and activate O2generation.
In addition to lectins or mucus, simple agitation seems to influence the glycocalyx.
Matsusato and Kobayashi [
171
] argued that Chattonella-mediated fish mortality could be
due to inhibition of respiratory water flow through the gills by mucus substance derived
from the flagellate cells. It has been reported that Chattonella can secret mucus substances
when the cells were passed through a net with 95
µ
m mesh size. Thus, it could be inferred
that mucous substances on the gill surface of fish exposed to Chattonella are at least partly
derived from Chattonella cells. Moreover, indirect immunofluorescence using antiserum
raised against crude glycocalyx of C. marina suggested the presence of glycocalyx, together
with C. marina cells on the gill surface of fish exposed to C. marina [232].
Ishimatsu et al. [
168
,
229
] demonstrated that the earliest physiological and histological
changes observed in the yellowtail after Chattonella exposure was a rapid drop of arterial
oxygen partial pressure and considerable accumulation of mucous substances between
the filaments and lamellae of the gill tissues, respectively. Based on the previous studies,
it seems obvious that glycocalyx plays a pivotal role in the ichthyotoxic mechanism of
Chattonella and other raphidophycean flagellates.
4. Raphidophycean Flagellates
Heterosigma akashiwo,Olisthodiscus luteus, and Fibrocapsa japonica often cause serious
mortality of wild and farmed fish [
267
]. Generation rates of superoxide anion (O
2
) and
hydrogen peroxide (H
2
O
2
) by C. marina (two strains), C. antiqua,H. akashiwo,O. luteus, and
F. japonica were estimated by SOD-inhibitable cytochrome c reduction and scopoletin assay,
respectively [
88
]. Chattonella showed the highest O
2
and H
2
O
2
production rates among
the raphidophytes tested, based on cell number. This may be due to different cell sizes.
Chattonella has nearly ten times larger cell size than other raphidophycean flagellates.
Antioxidants 2022,11, 206 11 of 28
Interestingly, an increase in H
2
O
2
levels of disrupted cell suspensions of these raphi-
dophytes even higher than intact flagellate cell suspensions was observed [
161
]. These
findings suggest that these raphidophytes have a certain intracellular compartment where
H
2
O
2
might be accumulated at high concentration, from which it is gradually released into
the medium during normal growth. The presence of intracellular compartment with high
H
2
O
2
concentration may be a common cellular feature of raphidophytes. Regarding the be-
havior of H
2
O
2
, it has been demonstrated that there was a decrease in the concentration of
exogenously added H
2
O
2
in C. marina cell suspension, with approximately 30 min half-life,
whereas H2O2in the culture medium alone was much more stable [161].
5. Cochlodinium Polykrikoides
Cochlodinium polykrikoides is a harmful dinoflagellate with potent fish-killing activ-
ity [
59
,
90
,
268
]. Apart from C. polykrikoides,C. fulvescens [
59
,
269
,
270
] and Cochlodinium sp.
Type Kasasa [
271
] have been identified as morphologically similar and toxic species. Cells
with 28–35
µ
m diameter form 4–8 chains, depending on growth conditions. Cochlodinium
blooms have been reported in Japan, Korea, and other countries [
272
,
273
], and cause fish
mortality [
269
,
274
]. For instance, C. polykrikoides bloom caused economic losses of more
than USD 100 million to fisheries in Korea [62,275].
Previous studies reported that certain toxic compounds, including neurotoxin, hemolytic
toxin, haemagglutinative agent, and paralytic shellfish poisoning (PSP) toxins were found
in C. polykrikoides [
3
,
58
,
61
,
276
]. Kim et al. reported that C. polykrikoides isolated in Korea
generated O
2
and H
2
O
2
[
62
] and proposed that C. polykrikoides exerts gill tissue damage
and fish mortality through ROS production [
277
]. Contrarily, C. polykrikoides isolated in
Japan produced O
2
and H
2
O
2
with much lower levels than those of Chattonella marina,
and the strains isolated in Japan did not respond to lectins and fish mucus [
178
]. Further
studies showed that only trace levels of ROS were detected in cell suspensions of five clonal
strains of C. polykrikoides isolated from different localities, and at times in Japan. To evaluate
the fish-killing activity of the Japanese strains of C. polykrikoides with low ROS generation
activity, damselfish (Chromis caerulea; average length 3
±
0.8 cm) were exposed to the
flagellates (
4×103cells/mL
), and the results showed that damselfish were susceptible to
all the strains of C. polykrikoides tested, with 100% mortality within 90 min of exposure,
whereas no significant protective effects of SOD and catalase were observed in the exposure
experiments [
194
]. The reason for the discrepancy between the Korean and the Japanese
strain of C. polykrikoides is still unclear. However, since ROS level is dependent on strain,
growth conditions, and assay methods [
88
,
173
,
184
], it is possible that the Korean strain
may have an extremely more potent ROS generation activity than the Japanese strains. In
addition, there is a possibility that certain toxic factors other than ROS might be mainly
involved in the ichthyotoxicity of C. polykrikoides. Regarding the fish-killing mechanism
of C. polykrikoides, suffocation caused by huge amount of mucus substances derived from
C. polykrikoides could be responsible for fish death [
61
,
276
]. Exposure experiments using
several fish species demonstrated that still-unknown toxic agents together with mucus
substances secreted from C. polykrikoides may be responsible for fish mortality [
269
]. Fur-
thermore, C. polykrikoides continuously secretes large amounts of mucous substances into
the medium as a characteristic feature [
178
]. Therefore, it could be inferred that the mu-
cus and certain toxins, including ROS, may be involved in the fish-killing mechanism
of C. polykrikoides. Interestingly, Shin et al. have reported that C. polykrikoides induced
oxidative damage and DNA degradation in the gill of red seabream after exposure to
sub-lethal concentrations of the flagellate [278].
6. Karenia Mikimotoi
The dinoflagellate Karenia mikimotoi (K. mikimotoi), formerly Gyrodinium aureolum,
G. cf. aureolum
,G. type-’65, G. nagasakiense, and G. mikimotoi, is highly toxic to both fish
and shellfish [
279
]. K. mikimotoi is an unarmored dinoflagellate with average cell size of
23–40
µ
m in diameter and flattened, with a characteristic swimming [
280
]. K. mikimotoi is a
Antioxidants 2022,11, 206 12 of 28
eurythermal and euryhaline organism, which can survive at temperature range of
4–31 C
and salinity at 9–31 [
281
,
282
]. Additionally, K. mikimotoi grows under light intensities
(10 to 1200
µ
mol/m
2
/s) and can assimilate different chemical forms of nitrogen and
phosphorous [53,283].
HABs of K. mikimotoi have occurred in Japanese waters [
15
,
284
], the
North Atlantic [53,285]
,
and other areas [
286
,
287
]. HABs of K. mikimotoi have caused massive mortality of fish [
15
]
and shellfish in Japan [
288
]. Since the mid-1960s, when K. mikimotoi bloom occurred in
Japan [
289
], mortality of various fish and invertebrate species caused by K. mikimotoi has
been reported in Europe, Australia, Japan, South America, and North Africa [3].
Regarding the toxic mechanisms of K. mikimotoi, it has been reported that K. mikimotoi
produce several toxic agents, such as low-molecular-weight hemolytic toxins [
290
293
],
cytotoxic polyethers [
294
,
295
], and ROS [
181
,
296
]. Matsuyama reported that G. mikimotoi
strongly inhibited the filtration rate of bivalves [
297
]. Sellem et al. demonstrated that
the 18:5n3 fatty acid produced by G. mikimotoi exhibited detrimental effects on sea urchin
(Paracentrotus lividus) [
298
]. Mitchell and Rodger reported that K. mikimotoi bloom was
associated with fish and shellfish mortality [299].
In 2012, large-scale HAB of K. mikimotoi (2
×
10
3
–1.18
×
10
5
cells/mL) caused mass
mortality of Japanese pufferfish (Takifugu rubripes) in Japan [
300
]. Exposure studies con-
firmed that NGU04, a strain of K. mikimotoi isolated from the HAB area, was toxic to fish
during the time (0.3–4 h) in cell-density-dependent manner (5
×
10
2
–1
×
10
4
cells/mL).
Interestingly, NGU04 produced extremely high levels of ROS, which were nearly equal
to the levels of C. marina measured at the same time [
183
]. ROS generation by C. marina
increases in response to extracellular stimuli, such as lectins [
191
,
218
]. After binding to the
cell surface carbohydrate moieties, lectins induce various cellular signaling pathways, lead-
ing to the enhancement of ROS generation in leukocytes [
301
,
302
]. C. marina might possess
such pathways regulating ROS generation in response to lectin stimuli, as found in leuko-
cytes. NGU04 also increased ROS levels in the presence of three lectins, which had different
saccharide specificity. Additionally, the lectin response profile of NGU04 differed from that
of C. marina. This may reflect differences in cell surface structures between raphidophycean
flagellate C. marina and dinoflagellate K. marina, especially the lectin binding sites [
183
].
Regarding O
2
and H
2
O
2
generation mechanisms, Hymenomonas carterae, a marine phyto-
plankton, produced extracellular H
2
O
2
without utilizing O
2
[
222
]. Similarly, fluorescence
microscopic observation of the NGU04, using ROS-specific fluorescence probes, indicated
that O2and H2O2are produced in different intracellular compartments [183].
The zooplankton Brachionus plicatilis (B.plicatilis) is highly susceptible to K. miki-
motoi [
303
], with NGU04 exhibiting the most lethal effect against B.plicatilis among the
K. mikimotoi strains tested, whereas C. marina had no significant effect on the zooplankton
under the same experimental conditions [
183
]. Since the toxic potential of K. mikimotoi
on marine organisms, including shellfish, is well correlated with its toxicity against ro-
tifer [
303
,
304
], the response of rotifer to the algae could be a reliable assay for evaluating
K. mikimotoi toxicity. Moreover, a recent review [
305
] indicated that rotifer could be use-
ful for marine ecotoxicology studies. Thus, the results suggest that NGU04 can exert
potent toxicity on shellfish, as well as on fish. As supporting evidence for this, NGU04
demonstrated that it was lethal against juvenile abalone (Nordotis gigantea) in laboratory
experiments [
306
]. Furthermore, NGU04 exerted hemolytic activities against rabbit and
fish erythrocytes, and the activities were much stronger than other strains of K. mikimotoi
with different backgrounds [
306
]. Since antioxidant enzymes, such as SOD and catalase,
had no effect on rotifer toxicity of NGU04, it could be inferred that ROS might not be the
major toxic factor of the strain, at least against rotifer [
183
]. Therefore, it is possible that
K. mikimotoi exerts its toxic effect against rotifer and shellfish mainly through its hemolytic
activity. This notion is supported by the non-toxic effect of C. marina, with no hemolytic
activity against rotifer; however, Heterocapsa circularisquama with potent hemolytic activity
can kill rotifer and shellfish, but not fish.
Antioxidants 2022,11, 206 13 of 28
Overall, it could be concluded that K. mikimotoi, especially at high cell density blooms,
can negatively affect several surrounding organisms, not only through ROS-mediated
oxidative stress, but via its hemolytic activity.
7. Nitric Oxide (NO) Production in Marine Microalgae
Over the years, studies have shown that C. marina generates nitric oxide (NO) under
ordinal growth conditions [
185
]. As a first experiment, chemiluminescence (CL) reaction
between NO and luminol–H
2
O
2
was employed to detect NO in C. marina [
307
]. When H
2
O
2
and luminol were added to C. marina simultaneously, increased CL response was detected,
and it was significantly suppressed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-
1-oxyl-3-oxide (carboxy-PTIO), a specific NO scavenger [
308
]. Detailed kinetic analyses
indicated that NO production by C. marina was cell-density-dependent, and the level of
NO for 10
4
cells/mL was estimated to be nearly 10
µ
M. Griess reaction also confirmed the
NO production by C. marina [
309
]. Furthermore, NO was detected in C. marina cell sus-
pension using NO-reactive fluorescent probe diaminofluorescein-FM diacetate (DAF-FM
DA) technique; however, NO fluorescence was completely inhibited by carboxy-PTIO [
310
].
Moreover, fluorescence microscopic observation suggested that NO was generated intracel-
lularly in C. marina, and bright fluorescence of C. marina was inhibited by carboxy-PTIO. In
addition, a comparative study showed that significantly higher fluorescence was detected
in raphidophytes Chattonella ovata and Heterosigma akashiwo, whereas only trace levels of
fluorescence were observed in dinophytes Alexandrium tamarense,A. taylori,Cochlodinium
polykrikoides,Gymnodinium impudicum, and eustigmatophycean Nannochloropsis oculata.
These results suggest that relatively high level of NO production may be a common specific
feature of the raphidophycean flagellates [
204
]. To analyze the mechanism of NO produc-
tion in C. marina, the effect of an inhibitor of NO synthase (NOS), NG-Nitro-L-arginine
methyl ester (L-NAME) was examined. L-NAME is known to block NO production in
mouse macrophage cell line RAW264.7 cells [
311
]. Similarly, L-NAME inhibited NO pro-
duction by C. marina. In contrast, the addition of L-arginine, a substrate for NOS, resulted
in an increase in NO level, with results obtained by luminol–H2O2assay.
NO, a gaseous free radical initially described as an endothelium-derived relaxing
factor [
312
], is involved in various biological processes in mammals [
186
]. NO plays nu-
merous roles not only in animals but also in plants. NO is membrane permeable and is
involved in many important processes in plants [
187
189
]. Apart from higher plants, green
algae and cyanobacteria also produce NO [
313
315
]. Similarly to C. marina, some species of
marine microalgae also generate NO under certain conditions [
102
,
316
,
317
]. The results
obtained by three independent assay methods demonstrated that C. marina is capable
of producing NO without specific stimuli or stress conditions. In mammalian systems,
NO is mainly produced from L-arginine by NOS, which yields L-citrulline and NO [
318
].
In plants, nitrate reductase (NR) has been demonstrated as a major NO generation sys-
tem [
319
]. Moreover, NO is also produced by non-enzymatic nitrite reduction at acidic
conditions [
320
]. In contrast to C. marina, it has been reported that unicellular alga Chlamy-
domonas reinhardtii produces NO through NR activity [
315
], and not through the activity
of NOS-like enzyme. As observed in C. reinhardtii, NO production by Chlorella sorokiniana
occurred in darkness and required nitrite [
321
]. Furthermore, it has been suggested that the
NR-mediated NO production activity of these microalgae was linked with photosynthetic
electron transport system because illumination of the algae cells suppresses NO production;
however, the suppressive effect can be reversed by 3,4-dichlorophenyl-1,1-dimethylurea,
a photosynthesis inhibitor [
315
]. Thus, it is likely that these unicellular algae may have a
common NO production mechanism that can be affected by various growth conditions.
Regarding C. marina, continuous NO production was observed under normal growth
conditions (under illumination), and exogenous nitrite had no effect, suggesting that NR
may not be involved in NO generation. Similarly to C. marina, some evidence suggests
the presence of NOS-like activities in photosynthetic organisms [
316
,
322
]. Furthermore, a
pathogen-induced NO-synthesizing enzyme has been purified from tobacco leaves [
323
].
Antioxidants 2022,11, 206 14 of 28
Such pathogen-induced NO production in plants may play an important role in defense
mechanism against pathogens through hypersensitive response [189].
NO has been shown to be an important regulator of mucus secretion in the stom-
ach [
324
326
], and NO donors induced mucus secretion from isolated gastric mucus
cells [
325
]. Therefore, it is conceivable that NO alone or in combination with ROS may
induce over-secretion of mucus substances on the gill surface of fish exposed to C. marina
cells, leading to the blockage of respiratory water flow. Shimada et al. [
327
] observed the
presence of highly concentrated NOx in the cortex of Chattonella antiqua, and they proposed
that NOx may induce mucous discharge from gill surface when C. antiqua cells pass be-
tween gill lamellae. NO [
328
] and its oxidized stable product, nitrite [
329
], are also known
to oxidize hemoglobin to methemoglobin that cannot transport oxygen, leading to tissue
hypoxia [
330
]. Regarding histological findings in the gills of fish exposed to C. marina, it
has been reported that C. marina induced mucus secretion and altered gill lamella integrity
in goldlined seabream after exposure, but no significant increase in methemoglobin was
observed in the fish, even after developing symptoms [331].
Apart from the possible factor involved in fish-killing mechanism of C. marina, NO
concentration of marine environments is 10
4
times higher than atmospheric NO level due
to extensive photolysis of nitrate and nitrification processes [
332
], and some species of
microalgae release NO during the natural growth process [
102
,
317
]. Overall, these findings
suggest that NO could be a stressor of marine organisms in multiple ways.
For a quick overview of this review, the most harmful and notable HAB-forming
species, together with some topics specific to the species, are summarized in Table 4.C. ma-
rina,C. antiqua,C. polykrikoides, and Karenia mikimotoi are well-recognized ROS-producing
harmful algae, and ROS seem to play pivotal roles in the ichthyotoxic mechanisms, while
hemolysin might be responsible for shellfish toxicity.
Table 4. The most harmful and notable HAB-forming species highlighted in this review.
Species Main Toxic Factors Detected Susceptible Organisms Topics
Chattonella
marina/antiqua
(Raphidophyte)
1 ROS (superoxide,
hydrogen peroxide, and
hydroxyl radical)
2 Nitric oxide (NO)
Fish
1 NADPH oxidase is proposed as a
mechanism of ROS production, which
might be located on glycocalyx, a cell
surface structure [94,162,193,219].
2 The highest ROS generation rate
among the species tested so far [184].
Cochlodinium polykrikoides
(Dinoflagellate)
1 Hemolysin
2 ROS (superoxide and
hydrogen peroxide)
Fish
Shellfish
1 Secretion of huge amount of
highly viscous mucus-like substances
[61,276].
Karenia mikimotoi
(Dinoflagellate)
1 Hemolysin
2 ROS (superoxide and
hydrogen peroxide)
Fish
Shellfish
1 Extremely toxic to both fish and
shellfish, and HABs due to this
dinoflagellate are often associated
with mass mortality of both fish and
shellfish [279,299].
8. Conclusions
HABs are a serious threat to marine resources and fisheries. Anthropogenic changes,
including global warming, can further increase the distribution of HABs and the appearance
of new HAB species. However, studies are yet to comprehensively elucidate the toxic
mechanism of HAB species. In this review, we focused on raphidophytes (Chattonella
marina,C. antiqua, and Heterosigma akashiwo) and dinoflagellates (Karenia mikimotoi and
Cochlodinium polykrikoides), which are the major groups of HAB species. Since HABs of
these species frequently cause mortality of wild and farmed marine organisms, with huge
economic losses (Table 4), it is necessary to have a comprehensive understanding of the
HAB species, their toxic mechanisms, and their blooming period or conditions, which may
help in minimizing their impact.
Antioxidants 2022,11, 206 15 of 28
The findings of this review showed that most of the harmful algae, especially the
ichthyotoxic species described above, produced relatively higher levels of ROS, with
Chattonella having the highest production rate. Although extensive studies are required to
fully understand the biological significances of ROS production by HAB species and their
impact on surrounding ecosystems and organisms, it is probable that ROS play pivotal
roles in the fish-killing activities of HAB species, such as Chattonella, which is supported by
the findings of previous studies. ROS production alone may not sufficiently explain the
ichthyotoxic mechanism of the HAB species and could be attributed to synergistic effects of
multiple factors. Therefore, additional studies are needed for a comprehensive elucidation
of the synergistic effects of multiple factors in the fish-killing activities of flagellate cells.
For instance, the biochemical and cellular structural characteristics of Chattonella and
the physiological vulnerability of gill tissue of susceptible fish species to the flagellates
should be examined. Although studies have shown that Chattonella extracts possess several
bioactive compounds with hemolytic [
3
,
333
] and antioxidant [
334
337
] activities, their
biological significance is still an open question. Further efforts can help elucidate the exact
roles of ROS and other bioactive molecules and the detailed processes leading to eventual
fish death. Overall, current findings on Chattonella are summarized in a schematic diagram
(Figure 1).
Antioxidants 2022, 11, x FOR PEER REVIEW 16 of 28
(Chattonella marina, C. antiqua, and Heterosigma akashiwo) and dinoflagellates (Karenia
mikimotoi and Cochlodinium polykrikoides), which are the major groups of HAB species.
Since HABs of these species frequently cause mortality of wild and farmed marine
organisms, with huge economic losses (Table 4), it is necessary to have a comprehensive
understanding of the HAB species, their toxic mechanisms, and their blooming period or
conditions, which may help in minimizing their impact.
The findings of this review showed that most of the harmful algae, especially the
ichthyotoxic species described above, produced relatively higher levels of ROS, with
Chattonella having the highest production rate. Although extensive studies are required
to fully understand the biological significances of ROS production by HAB species and
their impact on surrounding ecosystems and organisms, it is probable that ROS play
pivotal roles in the fish-killing activities of HAB species, such as Chattonella, which is
supported by the findings of previous studies. ROS production alone may not sufficiently
explain the ichthyotoxic mechanism of the HAB species and could be attributed to
synergistic effects of multiple factors. Therefore, additional studies are needed for a
comprehensive elucidation of the synergistic effects of multiple factors in the fish-killing
activities of flagellate cells. For instance, the biochemical and cellular structural
characteristics of Chattonella and the physiological vulnerability of gill tissue of
susceptible fish species to the flagellates should be examined. Although studies have
shown that Chattonella extracts possess several bioactive compounds with hemolytic [3,333]
and antioxidant [334337] activities, their biological significance is still an open question.
Further efforts can help elucidate the exact roles of ROS and other bioactive molecules
and the detailed processes leading to eventual fish death. Overall, current findings on
Chattonella are summarized in a schematic diagram (Figure 1).
Figure 1. Production of reactive oxygen species (ROS) and other bioactive molecules in Chattonella.
Author Contributions: K.C. coordinately wrote the manuscript with T.O. and designed the
construction of the review. K.C. collected information and arranged all tables. M.U. and Y.L.
searched and arranged all the references. M.U. and Y.L. also provided important opinions
regarding the completed manuscript. D.K. provided funding for the research. T.O. and D.K.
provided major ideas for the design of the manuscript and finally reviewed the completed
manuscript. All authors have read and agreed to the published version of the manuscript.
Figure 1. Production of reactive oxygen species (ROS) and other bioactive molecules in Chattonella.
Author Contributions:
K.C. coordinately wrote the manuscript with T.O. and designed the construc-
tion of the review. K.C. collected information and arranged all tables. M.U. and Y.L. searched and
arranged all the references. M.U. and Y.L. also provided important opinions regarding the completed
manuscript. D.K. provided funding for the research. T.O. and D.K. provided major ideas for the
design of the manuscript and finally reviewed the completed manuscript. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by grants from the National Marine Biodiversity Institute of Korea
(MABIK 2022M00100, and 2022M00300) and by the National Research Foundation of Korea (NRF-
2017R1A2B4005582). This work was also supported in part by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18K05823).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Antioxidants 2022,11, 206 16 of 28
Data Availability Statement:
The datasets used and analyzed during the current study are available
from the corresponding author on reasonable request..
Conflicts of Interest: The authors declare no conflict of interest.
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