Identification of toxic and non-toxic Microcystis by three primers

To discriminate from toxic and non-toxic Microcystis, 3 pairs of primers epF/mb1R, mcF/teR and mcF/umR were designed, based on sequences of microcystin synthetase gene cluster. The primers were applied to test the toxicity of 19 strains of Microcystis, which came from different places, including China, America, Japan and Australia, via whole-cell PCR. All these primers, cpF/mb1R, mcF/teR and mcF/umR, were able to amplify the expected bands from 15 toxic strains of Microcystis. The amplified fragments were 2,800bp, 1,800bp, 2,300bp respectively. The bands were proved to be the fragments of microcystin synthetase gene cluster according to blasting the sequences datum of PCR products on NCBI. In addition, we compared the results by two methods of PCR and HPLC. It showed that both PCR and HPLC methods gave a similar result on toxicity of Microcystis strains, and indicated that 3 pairs of primers could distinguish between toxic and non-toxic Microcystis. To detect the sensitivity of the primers, the threshold of primers for cell concentration was also studied. The results showed that the optimal concentrations were from 2 x 105 cells/mL to I X 107 cells/mL. Especially, the primes mb1R/epF, mcF/umR could amplify the expected bands on the cells concentrations of from 2 X 104 cells/mL. It was concluded the 3 primers were highly sensitive,and able to apply for discrimination of toxic and non-toxic strains of Microcystis

ANALYSIS OF OFF-FLAVORS IN FISH BY MICROWAVE MEDIATED DISTILLATION WITH HEADSPACE SOLID-PHASE MICROEXTRACTION AND GAS CHROMATOGRAPHY-MASS SPECTRUM

Aquaculture the rearing of fish and other aquatic animals is the best approach for meeting the increasing demand for aquaculture products worldwide. However, the problem of off-flavor has been documented as a serious setback to aquaculture. Though there are many off-flavors can result in a fish being objectionable, approximately 80% of the rejected fish have the earthy-musty odor, which is primary caused by the presence of either 2-methylisoborneol (2-MIB) or geosmin. Based on the fact that the detection of earthy-musty odorous compounds in fish mainly relied on the sensory method in China, the present study established the instrumental method to detect 2-MIB and geosmin by microwave mediated distillation with headspace solid-phase microextraction and gas chromatography-mass spectrum (MWDE-SPME-GC-MS). Through investigating the effects of the key parameters, microwave processing time and carrier gas flow rate, in microwave mediated distillation on extracts, it is concluded that 6 ruin of microwave time and 70 mL/min nitrogen flow were the optimized condition on which earthy-musty odorous compounds could be completely distilled from fish flesh. Then the earthy-musty odorous compounds were absorbed in the fiber under HS-SPME, and were finally desorbed at 250 degrees C and determined by GC-MS. The limits of detection for 2-MIB and geosmin in fish were both 0.1 mu g/kg. There were significant linear correlation in the range of 1-20 mu g/kg for the two earthy-musty odorous compounds, with calibration coefficients at 0.987 and 0.995, respectively. Therefore, trace levels of earthy-musty odorous compounds at ppb in fish could be quantified by this method with credible results

EFFECTS OF COPPER SULFATE TREATMENT ON EUTROPHIC URBAN LAKE PHYTOPLANKTON COMMUNITIES

Copper sulphate treatment is widely used as a global and empirical method to remove or control phytoplankton blooms. We tested the acute toxicity of 5 species of familiar microalgae and gave an urban lake copper sulfate treat in order to illuminate how the water body response to copper treat, especially the change of phytoplankton species and density of microcystins. According to OECD Alga growth inhibition test, effect of copper on 5 species of familiar microalgae was tested, and species of Cyanophyta showed much higher sensitivity to copper than species of Chlorophyta and Bacillariophyta. In a eutrophic urban lake, CuSO4 center dot 5H(2)O was used as algicide to control the water bloom caused by cyanobacterial. The copper concentration applied was 102 mu g/L ( as copper). We investigated the lake response to copper sulfate I which showed that the transparence was substantially improved; TN and TP did not change a lot; total algal decreased just after the copper sulfate treatment, and later increased again; at the beginning of the experiment, species of Cyanophyta died and the amount sharply decreased, Bacillariophyta and Chlorophyta turned to be the preponderant species, which could be related to species sensitivity to copper, and species of Cyanophyta began to grow again and took the predominance few days later; density of microcystins in the water rapidly decreased to level lower than that in the treatment before, only in 4d

Establishment of cell model for the study of cytotoxicity induced by microcystin-LR

Microcystins are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These toxins cause hepatocyte damage by inhibiting protein phosphastases I and 2A, resulting in hyperphosphorylation of a number of different proteins. Furthermore, high incidence of primary liver cancer in Qidong and Haimen in China has been associated with bloom contaminated drinking water. The mechanisms of MC-induced hepatotoxicity and tumor promotion activity have not been fully elucidated. In this study, Using 18 cell lines as materials, the cytotoxicity induced by different concentrations of microcystin-LR (20 and 50 mu g/mL) was studied. The morphological observation showed that five cell lines (PC-3, J82, 786-O, 5637 and VERO-E6) underwent obviously morphological changes after 30h treatment with microcystin-LR ( MCLR) with a dose-dependent manner. The cytoxicity of MCLR was determined by LDH leakage that showed a dose-dependent increase in five cell lines treated with MCLR, of which LDH leakage for 5637 and PC-3 were more serious under the same treatment. The SOD activity for five cell lines tended to increase in 20 mu g/mL MCLR treatment but sharply decrease in 50 mu g/mL MCLR treatment compared with the control cells; the GSH content decreased totally in different treatment. Based on the analysis of the sensitivity discrepancy for MCLR, 5637 cell lines was chosen for the establishment of a cell model for the study of cytotoxicity induced by microcystin-LR

The spatio-temporal distribution of algal biomass in Dianchi Lake and its impact factors

Dianchi Lake is a typical plateau, unstratificated, eutrophic lake in southwest of China. It has been seriously polluted in the past decades. And it had not been investigated in the whole lake for phytoplankton since 1980s. Thus, a project was carried out to find out what happened to the phytoplankton biomass and its regime dominance in the lake now.In this study, Chlorophyll a (Chl. a) had been monthly investigated according to the national standard method at 40 sites in Dianchi Lake from September, 2001 to August, 2002. Cell density and phytoplankton species composition had been investigated bimonthly as well. The results showed that Chl. a increased from 0.015mg/L to 0.10mg/L (monthly average values) during January to August, 2002. Correspondingly, water temperature increased from 10 degrees C to 28 degrees C. Chlorophyll concentrations of the south sites were lower than the north ones. Site one had the highest chlorophyll a value in all sites. We concluded that the southwester prevailing over the Dianchi Lake played a very important role. The horizontal movement of phytoplankton should be wind-induced. The water blooms mainly composed by cyanobacterial species. The percentage sometimes reaches to 100%. And Microcystis aeruginosa was the most common species. But in the spring of 2002, the dominance species was changed. Aphanizomenon spp. became dominant (69%) in March. It showed an obvious evidence for the seasonal succession. Moreover, the investigation indicated that the Pearson Correlation were high significant between chlorophyll a and water temperature (0.736, p < 0.01), total nitrogen (0.970, p < 0.01) and chemical oxygen demand (0.929, p < 0.01). Therefore, we concluded that phosphor-us was no longer the limited nutrition factor in Dianchi Lake to phytoplankton growth, nitrogen has taken the place. CODMn which is considered the token of organism population level, become the main chemical factor affecting the algal biomass. All the results indicate that Dianchi Lake is extremely eutrophication now; the blooms can last for 9-10 months in the year; the water is polluted seriously, especially the organic pollution, and it became an important factor which could affect the phytoplankton biomass directly; the species are simplex I Microcystis, Anabaena and Aphanizouwnon were the most popular bloom-forming cyanobacteria

Comparative studies on physiological responses at phosphorus stress of three waterbloom-forming cyanobacteria

In this paper, the physiological responses of the three waterbloom-forming cyanobacteria (Microcystis aerugillosa FACHB469, Anabaena flos-aquae FACHB245 and Oscillatoria planctonica FACHB708) at Phosphorus limitation in stationary cultures were studied. The phosphorus uptake rate is hyperbolic correlated to the extracellular nutrient concentration as described by the Michaelis-Menten equation. K for phosphorus of M. aeruginosa, A. flos-aquae and O. planctonica was 8.260, 2.199 and 4.926, respectively, which indicated that the tolerant ability to the phosphorus stress was in the decreasing order of A. flos-aquae, O. planktonica and M. aeruginosa in the phosphorus deficient condition. The capacity to luxuriously uptake phosphorus of M. aeruginosa was higher than that of O. planctonica and A. flos-aquae. This result was consistent with the phosphorus uptake kinetics experiments of the three cyanobacteria. Alkaline phosphatase acitivity (APA) of cyanobacteria were inversely correlated with external phosphorus concentration. The APA was obviously promoted under phosphorus deficiency stress. The capacity of excreting A P in the three cyanobacteria was significantly different under the same phosphorus limited conditions ( A. flos-aquae > O. planctonica > M. aeruginosa). Extracellular APA of A. flos-aquae was 10 and 5 times higher than that of M. aeruginosa and O. planctonica, respectively, when they were cultured in phosphorus deficient condition.The studies compared phosphorus uptake kinetics and capacity of excreting AP in the three cyanobacteria, which would help to understand the population succession of cyanobacteria in the environmental waterbodies

COMPARISON BETWEEN THE TOXIC MICROCYTIS SPECIES AND NON-TOXIC MICROCYSTIS SPECIES ORIGINATED FROM THE COCULTURE OF MICROCYSTIS AND POTERIOOCHROMOAS SP.

Predation of Poterioochromonas sp. on prey Microcystis is normally a one way process, i.e., prey cells were continuously ingested and gradually disappeared. However, it was not always the same. Occasionally, a patch of blue-green colored cells may re-appear from the bottom of the culture flask and gradually proliferated. Based on this phenomenon, experiments were designed by using three strains of Microcystis-M. aeruginosa AC, DS and PCC 7820-as prey organisms co-cultured with predator Poterioochromonas sp., respectively. After one or two months, eventually "transformed" Microcystis populations were obtained separately from each of co-cultured treatment. Nest PCR and 16S rDNA analysis identified these transformed cells to be Microcystis sp.. Furthermore, it was astonishingly found that these three transformed Microcystis populations had lost the microcystin-producing ability. The result strongly suggested that Poterioochromonas sp. played an important role in transforming the toxicity of Microcystis during predating process.Transformed, non-toxic Microcystis had higher value of caroteinoids/chlorophyll ratio but lower value of cyanophycin/chlorophyll ratio compared with original toxic strains. Light response curves indicated that the PS If quantum yield and photosynthetic activity of these non-toxic strains were higher than that of original toxic strains, moreover, non-toxic strains achieved the maximum photosynthetic activity at lower light intensity than that of toxic strains. This might explain why the non-toxic transformed strains outgrew the toxic wild strains in Microcystis and Poteroochromonas co-culture