Coupling of cylindrospermopsin and pho-harboring Verrucomicrobia supports the formation of Raphidiopsis blooms in low-phosphorus waters.

Cylindrospermopsin (CYN) can induce phytoplankton community to secrete alkaline phosphatase (ALP), which is one of the important strategies for the bloom-forming cyanobacterium Raphidiopsis to thrive in extremely low-phosphorus (P) waters. However, how bacterioplankton community, another major contributor to ALPs in waters, couples to Raphidiopsis through CYN, and the role of this coupling in supporting the dominance of Raphidiopsis in nature remain largely unknown. Here, we conducted microcosm experiments to address this knowledge gap, using a combination of differential filtration-based and metagenomics-based methods to identify the sources of ALPs. We found that, compared with algal-derived ALPs, bacteria-derived ALPs exhibited a more pronounced and sensitive response to CYN. This response to CYN was enhanced under low-P conditions. Interestingly, we found that Verrucomicrobia made the largest contribution to the total abundance of pho genes, which encode ALPs. Having high gene abundance of the CYN-sensing PI3K-AKT signaling pathway, Verrucomicrobia's proportion increased with higher concentrations of CYN under low-P conditions, thereby explaining the observed increase in pho gene abundance. Compared with other cyanobacterial genera, Raphidiopsis had a higher abundance of the pst gene. This suggests that Raphidiopsis exhibited a greater capacity to uptake the inorganic P generated by ALPs secreted by other organisms. Overall, our results reveal the mechanism of CYN-induced ALP secretion and its impact on planktonic P-cycling, and provide valuable insights into the role of CYN in supporting the formation of Raphidiopsis blooms.

Chlorella pyrenoidosa mitigated the negative effect of cylindrospermopsin-producing and non-cylindrospermopsin-producing Raphidiopsis raciborskii on Daphnia magna as a dietary supplement.

Feeding effects are crucial for evaluating the capacity of zooplankton to regulate phytoplankton populations within freshwater ecosystems. To examine the impact of the bloom-forming cyanobacteria Raphidiopsis raciborskii, which occurs in tropical and subtropical freshwaters, on the growth of zooplankton Daphnia in relation to toxins, filament length and fatty acid content, we fed D. magna with R. raciborskii only (cylindrospermopsin (CYN)-producing and non-CYN-producing, as the negative controls), Chlorella pyrenoidosa only (as the positive control) and a mixed diet containing R. raciborskii (CYN-producing and non-CYN-producing) and C. pyrenoidosa. Consequently, our findings revealed that the toxic effect of CYN-producing R. raciborskii strains on Daphnia was mitigated by the coexistence of C. pyrenoidosa containing stearidonic acid (SDA, C18:4 ω3) in mixed diets. This was evident in the elevated survival rate compared that from diets containing only R. raciborskii and a significantly higher reproduction and population intrinsic increase rate compared to diets consisting of only R. raciborskii or C. pyrenoidos. Additionally, a strong positive correlation was observed between arachidonic acid (ARA, 20:4ω6) and the population intrinsic increase rate of Daphnia; notably, R. raciborskii strains were found to be rich in the ω6 polyunsaturated fatty acid ARA. These outcomes reinforce the crucial role of polyunsaturated fatty acids in predicting the population increase of crustacean zooplankton, which has long been neglected. Furthermore, our results underscore the potential effectiveness of zooplankton, particularly in temperate lakes, in controlling CYN-producing R. raciborskii populations.

Iron enrichment from hypoxic hypolimnion supports the blooming of Raphidiopsis raciborskii in a tropical reservoir.

Occurring worldwide, blooms of Raphidiopsis raciborskii threaten the use of water resources especially in tropical and subtropical waterbodies. Its high flexibility in the uses of light and macronutrients (C, N, P) frustrates any bloom prediction and control based on macronutrients regulation. To identify the critical factors promoting periodic blooms of R. raciborskii, the trends of meteorological, hydrodynamic, physical, and chemical variables (including macro- and micronutrients: N, P, Fe) were analyzed in a Chinese tropical large reservoir (Dashahe reservoir) over five years. It was hypothesized that Fe availability, mediated by the mixing pattern of the reservoir, played a crucial role in the periodic blooms of the cyanobacterium. To have a more complete understanding, the effects of Fe on growth of a local R. raciborskii strain were tested in a monoculture experiment. The biomass and relative abundance of R. raciborskii in the reservoir showed a clear seasonal trend, with relative abundance > 50% in summer/autumn (July to October). Three habitat types along a dominance gradient were identified in the reservoir and 17 variables were used to compare them. Statistical analysis and habitat comparison showed that temperature and stratification, dissolved Fe and N concentrations in the epilimnion, and dissolved Fe and oxygen concentrations in the hypolimnion were the critical factors driving the dynamics of R. raciborskii in the study reservoir. The habitat dominated by R. raciborskii was characterized by a relatively low availability of macro resources (Zeu/Zm < 1, SRP < 0.01 mg/L, DIN < 0.3 mg/L) and by a high Fe availability supplemented from hypoxic hypolimnion. The dependence of growth on Fe concentration increase was confirmed in culture where the maximum was reached at 0.689 mg Fe /L. Our results suggest that a high Fe bioavailability, also originating from the hypoxic hypolimnion, influences the dynamics R. raciborskii and favors the blooms of the species. As a consequence, Fe concentrations in the water column as well as oxygen measurements along the water column should be routinely included in the monitoring programs aimed at predicting and controlling R. raciborskii blooms.

Phosphorus deficiency stimulates dominance of Cylindrospermopsis through facilitating cylindrospermopsin-induced alkaline phosphatase secretion: Integrating field and laboratory-based evidences.

Potentially toxic Cylindrospermopsis raciborskii blooms are of emerging concerns, as its scale is spreading from tropical regions to high latitudes, increasing the risk of aquatic biota being exposed to cylindrospermopsin (CYN). So far, CYN-producing C. raciborskii strains have only been reported in tropical waters which are commonly phosphorus (P)-deficient, where they can dominate phytoplankton communities. However, the influence of CYN on phytoplankton communities under different P status remains unclear. In this study, we first analyzed the summer observations of 120 tropical reservoirs in Guangdong Province. The proportion of potential CYN-producers was significantly higher in P-deficient and CYN-present reservoirs than that in P-sufficient or CYN-absent ones. This suggested that in P-deficient condition, the potential CYN producers might gain more advantages by the help of CYN. Then, in laboratory experiments we found that upon P deprivation, CYN did not inhibit the cell growth of other algal cells, but significantly stimulates them to secret more alkaline phosphatase (ALP) than in P-sufficient condition. Through transcriptomics, we further revealed that under such P-deficient condition, CYN remarkably induced intracellular nitrogen allocation and protein export system by activating the PIK3/Akt-cGMP/PKG signaling pathways in Scenedesmus bijugatus, thus enhancing its ALP secretion. Our study implies that CYN-induced ALP secretion is facilitated upon P deficiency, thus supporting the dominance of its producers C. raciborskii.

[Intraspecific Variation in Growth and Alkaline Phosphatase Activity of Cylindrospermopsis raciborskii Strains in Response to Different Phosphorus Concentrations and Sources].

The cyanobacterial species C. raciborskii are ubiquitous in tropical regions, and its successful invasion into temperate zones has been partially attributed to its ability of survival in low P availability and the existence of multiple ecotypes. To explore the physiological response of different strains to phosphorus fluctuations, four strains of C. raciborskii isolated from the Zhenhai Reservoir were used to investigate their growth and alkaline phosphatase (ALP) activity at different inorganic phosphorus (Pi) concentrations (HP=7.13 mg ·L-1, MP=0.64 mg ·L-1, LP=0.03 mg ·L-1) and different phosphorus forms [dipotassium hydrogen phosphate (K2HPO4), sodium pyrophosphate (K4P2 O7), sodium polyphosphate (K5P3O10), D-glucose-6-phosphate (D-G-6-P), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP)]. Four C. raciborskii strains showed a similar growth response to phosphate changes: their biomass increased with an increase in Pi concentrations, while the ALP activity showed the opposite trend. The ALP activity of C. raciborskii N8 was significantly lower than that of other three strains, regardless of inorganic phosphorus concentrations, suggesting that this strain had a higher adaptability to phosphorus fluctuations. When cultured with different phosphorus forms, the biomass of C. raciborskii N8 and N9 in three dissolved inorganic phosphorus (DIP) compounds were significantly higher than those in three dissolved organic phosphorus (DOP) compounds, with the maximum and minimum specific growth rate in K2HPO4and ATP treatments, respectively. C. raciborskii preferred DIP although they can also utilize DOP to sustain its growth. Under the DOP conditions, the ALP activity of C. raciborskii N8 in the ATP treatment was significantly higher than that in the other two organic phosphorus compounds, while we did not observe similar results in C. raciborskii N9, indicating that strain N8 was more sensitive to DIP deficiency. Our results showed an intraspecific variation within C. raciborskii strains from the same reservoir. Compared with the other strains, strain N8 represented better adaptability to phosphorus fluctuations and DIP deficiency. Variations within C. raciborskii strains may make this species more adaptable to environmental changes and enhance its competitive advantage.

Do bigheaded carp act as a phosphorus source for phytoplankton in (sub)tropical Chinese reservoirs?

Stocking of bigheaded carp (mainly Hypophthalmichthys nobilis and H. molitrix) is commonly used in (sub)tropical Chinese reservoirs to control phytoplankton, but with ambiguous results. Whether these carp act as a phosphorus (P) source or sink for phytoplankton is debated. We compared the trophic structures in twenty-three reservoirs with different nutrient concentrations in the flood season (after bigheaded carp introduction) with the dry season (after bigheaded carp harvesting). Fish biomass was positively related to TP, and the slope of the relationship showed no difference between seasons. Bigheaded carp harvesting exceeded the amount introduced, which may explain an observed lower intercept of the relationship and fish biomass to the TP ratio in the dry season. Fish predation pressure on zooplankton (fish: zooplankton biomass ratio as a proxy) was highest in the flood season and increased with TP in both seasons. Accordingly, zooplankton grazing effect on phytoplankton (zooplankton: phytoplankton biomass ratio as a proxy) decreased with fish biomass. Furthermore, both the zooplankton biomass and the zooplankton: phytoplankton biomass ratio were among the lowest reported in the literature for the nutrient range studied. Fish grazing effect on phytoplankton (fish: phytoplankton biomass ratio as a proxy) was also highest in the flood season and decreased with TP in both seasons. Nanoplankton was the dominant phytoplankton group in oligotrophic to mesotrophic reservoirs, while filamentous cyanobacteria dominated in eutrophic reservoirs. Chlorophyll a increased with TP and fish biomass, whereas the yield of chlorophyll a per TP (Chla: TP ratio) increased with fish biomass. Accordingly, both chlorophyll a and the Chla: TP ratio were highest in the flood season. We conclude that bigheaded carp act as P sink at the ecosystem level but as P source for phytoplankton, and enhance the yield of chlorophyll a per TP and thus eutrophication.

Species diversity and seasonal dynamics of filamentous cyanobacteria in urban reservoirs for drinking water supply in tropical China.

Filamentous cyanobacteria have been observed to become the dominant species in reservoirs, especially in small reservoirs for drinking water supply in southern China. The occurrences of filamentous cyanobacteria blooms in such reservoirs add additional costs for water plants by decreasing the filtration efficiency and the potential of toxin production. To serve the purpose of drinking water supply, the effective risk assessment requires the dynamic pattern of filamentous cyanobacteria. This study seasonally collected samples from 25 reservoirs in Dongguan, one of the most important 'world factories' in China in July, December and March, and investigated the temporal dynamics of phytoplankton, particularly cyanobacteria community. Our investigation showed that filamentous cyanobacteria, Planktothrix sp, Limnothrix sp. and Cylindrospermopsis raciborskii dominated in these reservoirs and climate-related water temperature was the primary factor for the seasonal shift of filamentous cyanobacteria. High abundance of filamentous cyanobacteria occurred in the high water level period with increasing temperature but less relevant with nutrient conditions. Our study observed the seasonal dynamics of filamentous cyanobacteria in tropical urban reservoirs and highlighted the association between temperature and filamentous cyanobacteria. our data and analysis provided an evidence that increased temperature could increase the likelihood of frequency and intensity of filamentous cyanobacteria blooms. In the scenario of global warming, more frequent monitoring of filamentous cyanobacteria and the potential to produce toxin should be considered for water quality and reservoir management.

Cyanobacterial removal by a red soil-based flocculant and its effect on zooplankton: an experiment with deep enclosures in a tropical reservoir in China.

As one kind of cheap, environmentally-friendly and efficient treatment materials for direct control of cyanobacterial blooms, modified clays have been widely concerned. The present study evaluated cyanobaterial removal by a red soil-based flocculant (RSBF) with a large enclosure experiment in a tropical mesotrophic reservoir, in which phytoplankton community was dominated by Microcystis spp. and Anabaena spp. The flocculant was composed of red soil, chitosan and FeCl3. Twelve enclosures were used in the experiment: three replicates for each of one control and three treatments RSBF15 (15 mg FeCl3 l-1), RSBF25 (25 mg FeCl3 l-1), and RSBF35 (35 mg FeCl3 l-1). The results showed that the red soil-based flocculant can significantly remove cyanobacterial biomass and reduce concentrations of nutrients including total nitrogen, nitrate, ammonia, total phosphorus, and orthophosphate. Biomass of Microcystis spp. and Anabaena spp. was reduced more efficiently (95%) than other filamentous cyanobacteria (50%). In the RSBF15 treatment, phytoplankton biomass recovered to the level of the control group after 12 days and cyanobacteria quickly dominated. Phytoplankton biomass in the RSBF25 treatment also recovered after 12 days, but green algae co-dominated with cyanobacteria. A much later recovery of phytoplankton until the day of 28 was observed under RSBF35 treatment, and cyanobacteria did no longer dominate the phytoplankton community. The application of red soil-based flocculant greatly reduces zooplankton, especially rotifers, however, Copepods and Cladocera recovered fast. Generally, the red soil-based flocculant can be effective for urgent treatments at local scales in cyanobacteria dominating systems.

[Distribution and Factors Affecting Cylindrospermopsis raciborskii in Guangdong Reservoirs].

Cylindrospermopsis raciborskii originating from tropical and subtropical regions is potentially toxic and attracts much attention due to its extension to the global temperate zone in recent years. Based on historical data of 20 reservoirs with different trophic levels (dry season, wet season, and transitional season of 2010), this study focuses on the analysis of the occurrence and distribution of C. raciborskii in the Guangdong Province. Based on the results, C. raciborskii was found in 19 of the 20 reservoirs and its biomass ranges from 0.0001-39.740 mg·L-1 and accounts for 0.02%-97.07% of the total phytoplankton biomass. Both a notable spatial and seasonal distribution of C. raciborskii were observed. Its occurrence is higher in the western coastal area (77.78%) than in the Zhujiang Delta (66.67%) and northern coastal area (33.33%) and is relatively lower in the dry season (40%) compared with the rainy season (70%) and transition season (85%). The trophic level has a significant effect on the presence of C. raciborskii, which is notably higher in eutrophic reservoirs (81.48%) than in mesotrophic reservoirs (66.67%) and oligotrophic reservoirs (33.33%). The redundancy analysis shows that C. raciborskii biomass is positively correlated with total nitrogen (TN) and the trophic state index (TSI) and negatively correlated with dissolved inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the secchi depth (SD). Thus, C. raciborskii in Guangdong reservoirs may be promoted by environmental factors such as high nitrogen contents, low phosphorus concentration, and transparency.