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.

Cyanophycin accumulated under nitrogen-fluctuating and high-nitrogen conditions facilitates the persistent dominance and blooms of Raphidiopsis raciborskii in tropical waters.

Nutrient storage is considered a critical strategy for algal species to adapt to a fluctuating nutrient supply. Luxury phosphorus (P) uptake into storage of polyphosphate extends the duration of cyanobacterial dominance and their blooms under P deficiency. However, it is unclear whether nitrogen (N) storage in the form of cyanophycin supports persistent cyanobacterial dominance or blooms in the tropics where N deficiency commonly occurs in summer. In this study, we examined genes for cyanophycin synthesis and degradation in Raphidiopsis raciborskii, a widespread and dominant cyanobacterium in tropical waters; and detected the cyanophycin accumulation under fluctuating N concentrations and its ecological role in the population dynamics of the species. The genes for cyanophycin synthesis (cphA) and degradation (cphB) were highly conserved in 21 out of 23 Raphidiopsis strains. This suggested that the synthesis and degradation of cyanophycin are evolutionarily conserved to support the proliferation of R. raciborskii in N-fluctuating and/or deficient conditions. Isotope 15N-NaNO3 labeling experiments showed that R. raciborskii QDH7 always commenced to synthesize and accumulate cyanophycin under fluctuating N conditions, regardless of whether exogenous N was deficient. When the NO3--N concentration exceeded 1.2 mg L-1, R. raciborskii synthesized cyanophycin primarily through uptake of 15N-NaNO3. However, when the NO3--N concentration was below 1.0 mg L-1, cyanophycin-based N was derived from unlabeled N2, as evidenced by increased dinitrogenase activity. Cells grown under NO3--N < 1.0 mg L-1 had lower cyanophycin accumulation rates than cells grown under NO3--N > 1.2 mg L-1. Our field investigation in a large tropical reservoir underscored the association between cyanophycin content and the population dynamics of R. raciborskii. The cyanophycin content was high in N-sufficient (NO3--N > 0.45 mg L-1) periods, and decreased in N-deficient summer. In summer, R. raciborskii sustained a relatively high biomass and produced few heterocysts (< 1%). These findings indicated that cyanophycin-released N, rather than fixed N, supported persistent R. raciborskii blooms in N-deficient seasons. Our study suggests that the highly adaptive strategy in a N2-fixing cyanobacterial species makes mitigating its bloom more difficult than previously assumed.

Nutrient Regulation of Relative Dominance of Cylindrospermopsin-Producing and Non-cylindrospermopsin-Producing Raphidiopsis raciborskii.

Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) can produce cylindrospermopsin (CYN) which is of great concern due to its considerable toxicity to human and animals. Its CYN-producing (toxic) and non-CYN-producing (non-toxic) strains co-exist commonly in natural water bodies, while how their relative dominance is regulated has not been addressed. In this study, we combined field investigation with laboratory experiments to assessed the relationship between toxic and non-toxic R. raciborskii abundances under different nutrient levels. The rpoC1- and cyrJ-based qPCR was applied for quantifying total and toxic R. raciborskii abundances, respectively. The field survey showed that toxic R. raciborskii was detected in 97 of 115 reservoirs where its proportion ranged from 0.3% to 39.7% within the R. raciborskii population. Both total and toxic R. raciborskii abundances increased significantly with trophic level of these reservoirs, consistent with our monoculture and co-culture experiments showing in an increase in R. raciborskii growth with increasing nitrogen (N) or phosphorus (P) concentrations. In the monoculture experiments, growth rates of non-toxic and toxic strains from Australia or China were not significantly different under the same culture conditions. On the other hand, in the co-culture experiments, the toxic strains displayed a significantly faster growth than non-toxic strains under nutrient-replete conditions, resulting in an obvious shift toward the dominance by toxic strains from day 3 to the end of the experiments, regardless of the strain originating from Australia or China. The reverse was found under N- or P-limited conditions. Our results indicated that the toxic strains of R. raciborskii have a competitive advantage relative to the non-toxic strains in a more eutrophic world. In parallel to an increase in dominance, both toxic strains grown in the mixed population significantly increased CYN production under nutrient-replete conditions as compared to nutrient-limited conditions, suggesting that CYN may be of significance for ecological advantage of toxic R. raciborskii. These results highlight the importance of nutrient availability in regulating abundances and strain dominance of two genotypes of R. raciborskii. Our findings demonstrated that elevated nutrients would favor the growth of CYN-producing R. raciborskii and CYN production, leading to more blooms with higher toxicity at global scale.

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.

Interactive effect of water level and flushing rate on population dynamics of a harmful cyanobacterial species: Raphidiopsis raciborskii.

Changes in water level and flushing rate directly affect to a large extent the biomass of harmful cyanobacteria, and drive the shift of phytoplankton composition between cyanobacteria dominance/non-dominance in eutrophic waters. Here, we gave a theoretical formula describing the combinational effect of water level and flushing rate on cyanobacterial biomass in eutrophic and well-mixed waters. We also formulated an equation predicting the water level and flushing rate at which cyanobacteria become non-dominating in such water columns. The formulae were confronted with field observations of a low-light adapted cyanobacterium in a large coastal reservoir of southern China. Our formulae demonstrate that water level and flushing rate have an interactive effect on the equilibrium biomass of low-light adapted cyanobacteria in mixed and turbid waters. The formulae were well fitted to the field observation of Raphidiopsis raciborskii population in the reservoir during four dry seasons. In agreement with the theoretical analysis, multiple regression analysis also showed that the interaction between water level and flushing rate is able to interpret the variation of R. raciborskii biomass in the water column. The two formulae are applicable for predicting the response of low-light adapted cyanobacteria to local climate change. Our findings have practical significance in designing measures against the dominance of low light-adapted cyanobacteria in reservoirs.

[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.

Life-history responses of Daphnia sinensis simultaneously exposed to Microcystis aeruginosa and Cylindrospermopsis raciborskii.

Many cyanobacterial species co-occur commonly in a freshwater ecosystem and can be consumed simultaneously by zooplankton. Both Microcystis aeruginosa and Cylindrospermopsis raciborskii are the dominant species and coexist in eutrophic tropical waters, and they together are assumed to have exert a stronger effect on the life history traits of cladocerans than a single cyanobacterial species. In the present study, we tested the hypothesis with life-history experiments of Daphnia sienesis, a large cladoceran species in tropics. In the experiments, M. aeruginosa and C. raciborskii were used as a mixture of food with Chlorella pyrenoidosa for the experimental animals. D. sienesis showed excellent growth and survival on sole diets of C. pyrenoidosa (CP). By contrast, Daphnia's growth decreased significantly and reproduction was completely inhibited when cyanobacteria comprised 100% of the food offered. The supplementation of C. pyrenoidosa into cyanobacterial diets significantly decreased their harmful effects on Daphnia, who improved the life history traits with the reduction of cyanobacterial percentage in mixed foods, irrespective of cyanobacterial species. Compared with the cultures of D. sienesis fed with a single cyanobacteria species, the animals in the treatments fed both M.aeruginosa-FACHB469 (F469) and C. raciborskii N8 (N8) had a lower growth rate in all the proportion of C. pyrenoidosa (25% CP, 50% CP or 75% CP). The strongest synergistic inhibition by the two cyanobacterial strains was found in the treatments with the 25% CP (i.e., 25% CP + 37.5% F469 + 37.5% N8), and no animal survived to maturity and reproduced. Thus, the simultaneous exposure to the two cyanobacterial species should be taken into account in assessing the ecological risks of cyanobacterial blooms, since multiple cyanobacterial coexistence can result in strong synergistic inhibition on growth and reproduction of zooplankton.

Seasonal variation of microcystins and their accumulation in fish in two large shallow lakes of China.

Bioaccumulation of microcystins (MCs) has been widely observed in aquatic vertebrates and invertebrates, but its seasonal and specific variations remain unclear. In the present study, dissolved MCs in water, algal cell-bound MCs and muscle tissue MCs of nine fish species were investigated monthly in two of the largest shallow lakes in China: Lake Taihu and Lake Chaohu. The fish species were grouped as carnivorous, planktivorous, and omnivorous fish. Seasonal variations in dissolved and algal cell-bound MCs in water and MCs contents of fish hepatopancreas and muscle were investigated in the two lakes from 2009 to 2010. Dissolved MCs in water ranged from 0.35 to 2.56 µg l-1 in Lake Taihu and 0.16 to 2.45 µg l-1 in Lake Chaohu, and showed seasonally a unimodal distribution. Algal cell-bound MCs also showed a similar seasonal variation in both lakes, but dissolved MCs in water peaked about one month later than algal cell-bound MCs. The MCs content in the Fish muscle was higher MCs from October to December than in the other months. For most of the fish species, it exceeded the tolerable daily intake value established by the WHO. The averaged MCs content in the muscle of carnivorous, planktivorous, omnivorous fish was 48.2, 28.7 and 37.8 µg kg-1 in Lake Taihu, respectively, and 27.8, 18.6 and 20.4 µg kg-1 in Lake Chaohu. It was significantly higher in carnivorous fish than in planktivorous and omnivorous fish, indicating that carnivorous fish has a higher exposure risk to the local people when consuming the harvested fish. The average ratio of hepatopancreas to muscle MCs contents was 13.0, 25.2, 13.8 for carnivorous, planktivorous, omnivorous fishes in Lake Taihu, respectively, and 18.0, 24.9, 14.8 in Lake Chaohu. These ratio for planktivorous fish almost doubled that for carnivorous and omnivorous fish. High correlation of MC content in carnivorous, omnivorous and planktivorous fish indicates that MCs can be delivered along trophic levels in the food chains.

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.

Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies.

Cylindrospermopsin (CYN) is a cyanotoxin that is of particular concern for its potential toxicity to human and animal health and ecological consequences due to contamination of drinking water. The increasing emergence of CYN around the world has led to urgent development of rapid and high-throughput methods for its detection in water. In this study, a highly sensitive monoclonal antibody N8 was produced and characterized for CYN detection through the development of a direct competitive time-resolved fluorescence immunoassay (TRFIA). The newly developed TRFIA exhibited a typical sigmoidal response for CYN at concentrations of 0.01⁻100 ng mL−1, with a wide quantitative range between 0.1 and 50 ng mL−1. The detection limit of the method was calculated to be 0.02 ng mL−1, which is well below the guideline value of 1 μg L−1 and is sensitive enough to provide an early warning of the occurrence of CYN-producing cyanobacterial blooms. The newly developed TRFIA also displayed good precision and accuracy, as evidenced by low coefficients of variation (4.1⁻6.5%). Recoveries ranging from 92.6% to 108.8% were observed upon the analysis of CYN-spiked water samples. Moreover, comparison of the TRIFA with an ELISA kit through testing 76 water samples and 15 Cylindrospermopsis cultures yielded a correlation r² value of 0.963, implying that the novel immunoassay was reliable for the detection of CYN in water and algal samples.

[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.

Competition between toxic and non-toxic Microcystis aeruginosa and its ecological implication.

The frequency of toxic cyanobacterial blooms has increased in recent decades, but the factors that regulate the dominance of toxin-producing cyanobacteria over non-toxin-producing strains of one species are still obscure. This study examined the effects of temperature, light intensity, nitrate and phosphate on the dominance of MC-producing and non-MC-producing strains of Microcystis aeruginosa in monoculture and co-culture experiments. In the monoculture experiments, growth rates of the non-MC-producing strain were higher than those of the MC-producing strain under the same growth conditions. However, at the end of the co-culture experiments, the MC-producing strain became surprisingly dominant in all treatments except when treated with extreme low phosphate concentrations. Higher temperatures and nutrient levels can shift the dominance more quickly towards the toxic strain. The dominance may be explained by allelopathic interactions through allelochemicals and other secondary metabolites, but not MC. Environmental factors such as extremely low phosphate content may exert an indirect effect on strain dominance by changing the production of allelochemicals. Our findings highlight the complications in predicting competitive outcome for cyanobacterial strains in natural environments.

Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum.

OBJECTIVES: This study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. DESIGN AND METHODS: Lanthanum elements labeled antibody and streptavidin-biotin system were used in the "capture sandwich" format simultaneously. RESULTS: The working range of TRFIA for RV IgM was 2-80 AU/mL and for CMV IgM was 5-400 AU/mL. Intra- and inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. CONCLUSION: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy.

Occurrence and dominance of Cylindrospermopsis raciborskii and dissolved cylindrospermopsin in urban reservoirs used for drinking water supply, South China.

The tropical cyanobacterium Cylindrospermopsis raciborskii is of particular concern for its invasive characteristics and production of the toxin cylindrospermopsin (CYN). The present study represents the first attempt to determine the distribution of C. raciborskii and CYN in tropical China. The presence of C. raciborskii and CYN, as well as the composition of phytoplankton, was determined from a total of 86 samples from 25 urban reservoirs for drinking water supply in Dongguan City of South China. The presence of C. raciborskii was observed in 21 of the 25 reservoirs and confirmed that this species has been widely distributed in the investigated reservoirs. C. raciborskii accounted for between 0.1 and 90.3 % of the total phytoplankton biomass and contributed to the majority of the phytoplankton in some reservoirs such as Tangkengbian and Xiagongyan. Its biomass was negatively correlated with NO3 (-)-N concentration and Secchi depth. Dissolved CYN was detected in more than one-half of the reservoirs with concentrations up to 8.25 µg L(-1), and it positively correlated with C. raciborskii biomass. Dissolved microcystins (MCs) were detected in 12 of the 25 reservoirs with a maximum concentration 1.99 µg L(-1). Our data strongly suggest that C. raciborskii and CYN could be important health hazards in urban reservoirs of South China and that more data are needed for further assessment.

Survivals of D. galeata in sub-tropical reservoirs: harmful effects of toxic cyanobacteria in food source.

In the present study, we tested the hypothesis that massive occurrence of cyanobacteria as food source is one of the factors limiting the distribution of the zooplankton Daphnia galeata in the warm water reservoirs of South China. D. galeata was fed with Chlorella pyrenoidosa, 4 strains of cyanobacteria (three strains of Microcystis aeruginosa: MC1, MC2 and MC3, and one strain of Pseudoanabaena sp. :PA), a mixture of C. pyrenoidosa and cyanobacterial strains, and natural particles from the reservoir where the animal was initially collected. On medium and high food levels (1 and 1.5 mg C L(-1)) of C. pyrenoidosa, D. galeata showed a typical Type III of survivorship, and had a high intrinsic rate of population increase (0.37-0.41). On low food levels, the intrinsic rate of population increase declined but lifespan largely increased (84 days). When feeding with natural particles, the intrinsic rate of increase was evidently reduced (0.19), but the maximal body length (2 mm) was much longer than that of individuals observed in the fields implying that D. galeata was strongly preyed upon by size-selective predators. Applying a mixture of Chlorella and cyanobacteria, the strain and proportion of cyanobacteria significantly affected life history variables of the animal and its somatic growth. On the sole diet of each stain of cyanobacteria, D. galeata failed to reproduce. Microcystin and colonial morphology of Microcystis strains reduced clutch size and somatic growth rate. In comparison, the less negative effect of the strain MC3 indicates that the morphology was critical for actual ingestion of toxic cyanobacteria by the zooplanktons. Pseudoanabaena sp. had a short filament (15 µm), can be easily used as a food by D. galeata. Composition of phytoplankton community plays a significant role in survival and population sizes of D. galeata and massive occurrence of cyanobacterial blooms limits the distribution of D. galeata in the reservoirs of South China.

[One step time-resolved fluorescence immunoassay for direct estimation of microcystins].

A direct-competitive time-resolved fluorescence immunoassay (TRFIA) for microcystin detection was established, which was based on europium labeled MCLR-BSA conjugant and microtiter plates coated with anti-mouse IgG. The optimal dilution of europium labeled MCLR-BSA conjugant is 1/50 and most appropriate titration of anti-microcystin-LR (MCLR) monoclonal antibodies is 100 ng/mL. The standard curve under the optimal conditions shows that the quantitative range is from approximately 0.05 ng/mL to 10 ng/mL. The detection limit of the method is 0.02 ng/mL for MCLR, recoveries for microcystin (> 94%) in quantitative range is satisfactory.