Strain, cell density, and nutrient condition affect patterns of diurnal vertical migration and superoxide production in a red-tide alga.

A red tide occurs when cell densities of autotrophic microalgae and some heterotrophic protists increase dramatically and thereby change the color of the sea. Red tides sometimes have negative impacts on human activities, such as fisheries and tourism. Most red-tide flagellates display diurnal vertical migration (DVM) in which cells normally migrate upward during the day and downward at night. This behavior promotes active growth, due to the effective acquisition of nutrients and light, as well as population density increase and cell aggregation. However, the factors and their interactions influencing DVM remain to be clarified, such that no algorithm exists that can precisely simulate the DVM pattern and the development of a red tide in the field. Chattonella marina complex (hereafter Chattonella) is a representative microalga of harmful red tides and some previous studies has suggested that Chattonella's DVM plays important roles in development of a red tide. Chattonella can produce a large amount of superoxide (•O2 -), which is responsible for the regulation of various physiological processes as well as its toxicity against microorganisms and animals. In the present study, we examined the effects of strain, growth phase, cell density, and nutrient deficiency on the pattern of DVM. In addition, we also measured the •O2 - level in most experiments to assess the relationship between DVM and •O2 - production. Some strains displayed clear DVM, whereas others aggregated at the surface all day in a fixed condition. Strains' DVM patterns did not show a relationship with •O2 - production. Moreover, the DVM became less clear at high cell density and in nitrogen- or phosphorus-depleted conditions. Although a previous study reported that the •O2 - production rate increased during the light period and decreased during the dark period, regardless of cell density, the diurnal pattern of •O2 - became less clear at a higher cell density in a Chattonella strain used in the present study. Our findings indicate that DVM and •O2 - production by a Chattonella population composed of various strains can change across developmental phases and environmental conditions. This characteristic may produce adaptability in species and increase the chances of a massive population increase.

Current environmental status of the oyster farms on Lake Kamo in Japan; viral control of the harmful bloom of Heterocapsa circularisquama.

Lake Kamo is an enclosed, low-inflow estuary connected to the open sea that is famous for oyster farming in Japan. In the fall of 2009, this lake experienced its first bloom of the dinoflagellate Heterocapsa circularisquama, which selectively kills bivalve mollusks. This species has been detected exclusively in southwestern Japan. The completely unexpected outbreak of H. circularisquama in the northern region is believed to have been caused by the contamination of purchased seedlings with this species. The water quality and nutrient data collected by our group from July through October over the past 10 years revealed that the environment of Lake Kamo has not changed significantly. However, in the open water around Sado Island, where Lake Kamo is located, the water temperature has increased by 1.80 °C in the last 100 years, which is equivalent to 2-3-fold the world average. This has resulted in a rise in the sea level, which is expected to further deteriorate the water exchange between Lake Kamo and the open sea and low dissolved oxygen in the bottom layer of the Lake and the associated dissolution of nutrients from the bottom sediment. Therefore, seawater exchange has become insufficient and the lake has become nutrient rich, making it prone to the establishment of microorganisms, such as H. circularisquama, once they have been introduced. We developed a method to mitigate the damage caused by the bloom by spraying sediments containing the H. circularisquama RNA virus (HcRNAV), which infects H. circularisquama. After ∼10 years of performing various verification tests, including field trials, this method was used at the Lake in 2019. During the 2019 H. circularisquama growth season, a small amount of sediment containing HcRNAV was sprayed on the lake three times, which resulted in a decrease in H. circularisquama and an increase in HcRNAV, indicating that this method is effective in diminishing the bloom.

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes.

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•-) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•- production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•- production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.

Interannual variations in abundance and distribution of Chattonella cysts, and the relationship to population dynamics of vegetative cells in the Yatsushiro Sea, Japan.

The fish-killing raphidophytes Chattonella spp. have a resting cyst stage. To investigate the abundance and distribution of Chattonella cysts and determine their relationship to the population dynamics of vegetative cells, we conducted field observations from 2002 to 2017 in the Yatsushiro Sea, a semi-enclosed embayment in Japan, and analyzed the data including environmental conditions. Analysis of sediment sampled in the spring (mid-April to early June), shows that cysts are relatively abundant in the northern to middle area, where initial vegetative cells and large blooms are frequently detected. The maximum density of cysts was 616 cysts cm-3 in the northern area in 2016. Mean cyst abundance in the spring varied interannually, ranging from 5 to 138 cysts cm-3. A significant positive correlation between mean cyst abundance in the spring and maximum density of vegetative cells the preceding summer was seen, but no significant correlation was observed the following summer. The first detected date of vegetative cells (FDD) each year, which is likely related to cyst abundance and environmental conditions influencing cyst germination and/or growth characteristics of vegetative cells, also varied interannually from mid-April to early June. Regression analyses showed that FDD tended to be early when cyst abundance and bottom-water temperature were high. However, no significant correlation was observed between mean cyst abundance and bloom timing (the period from FDD to the occurrence date of the bloom), and bloom duration the following summer, as was the maximum density of vegetative cells. Instead, the timing and duration of blooms were correlated significantly with meteorological factors (e.g., solar radiation) for a month after FDD. The results suggest that cyst abundance reflecting the bloom magnitude of the preceding summer contributes to the timing of the appearance of vegetative cells in the year, but that bloom occurrence is likely to be controlled by the growth dynamics of vegetative cells through environmental conditions rather than by cyst abundance. The three distinct peaks in Chattonella cysts and vegetative cells from 2002 to 2017 correspond to the timings just after the El Niño. Large-scale atmospheric variability and its global teleconnection are possibly linked to long-term population dynamics of Chattonella in the area through local meteorological conditions and their life cycle.

Extracellular secretion of superoxide is regulated by photosynthetic electron transport in the noxious red-tide-forming raphidophyte Chattonella antiqua.

The raphidophyte Chattonella antiqua is a noxious red-tide-forming alga that harms fish culture and the aquatic environment. Chattonella antiqua produces and secretes superoxide anions (O2-), and excessive secretion of O2- into the water has been associated with fish mortality. It is known that strong light stimulates the production of O2- in Chattonella spp. but the mechanism of the light-induced production of O2- remains to be clarified. In the present study, we examined the effects of light on extracellular levels of O2- and photosynthesis in C. antiqua. Extracellular levels of O2- rose during growth under high-intensity light, and the level of O2- was correlated with the photosynthetic parameter qP, which reflects the rate of transport of electrons downstream of photosystem II. The production of O2- was inhibited in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic electron transport, suggesting that reducing power derived from electron transport might be required for the production of O2-. By contrast, the production of O2- was enhanced in the presence of glycolaldehyde, an inhibitor of the Calvin-Benson cycle, suggesting that the accumulation of NADPH might stimulate the production of O2-. Thus, it is likely that the production of O2- is regulated by photosynthesis in C. antiqua.

Development of a method to assess the ichthyotoxicity of the harmful marine microalgae Karenia spp. using gill cell cultures from red sea bream (Pagrus major ).

The present study reports the development of a method to investigate ichthyotoxicity of harmful marine microalgae using cultured red sea bream (Pagrus major) gill cells. The cultured gill cells formed adherent 1-2 layers on the bottom of the culture plate and could tolerate seawater exposure for 4 h without significant alteration in cell survival. The microalgae Karenia mikimotoi, Karenia papilionacea, K. papilionacea phylotype-I, and Heterosigma akashiwo were cultured, then directly exposed to gill cells. After K. mikimotoi and K. papilionacea phylotype-I exposure, live cell coverage was significantly lower than in the cells exposed to a seawater-based medium (control cells; P < 0.05). Toxicity of K. mikimotoi cells was weakened when cells were ruptured, and was almost inexistent when the algal cells were removed from the culture by filtration. Significant cytotoxicity was detected in the concentrated ruptured cells, and in the concentrated of ruptured cells after freezing and thawing though cytotoxicity was weakened; whereas, cytotoxicity almost disappeared after heat treatment. In addition, examination of the distribution of toxic substances from the ruptured cells showed that cytotoxicity mainly occurred in the fraction with the resuspended pellet after centrifugation at 3000×g.