Effects of light conditions on growth and defense compound contents of Datura inoxia and D. stramonium.

We examined the effects of light conditions on plant growth and production of defense compounds in the toxic species Datura inoxia and D. stramonium. Specifically, we investigated morphological and physiological traits, including the contents of nitrogen-based tropane alkaloids (atropine and scopolamine) as defense compounds, under three light conditions: 100%, 80%, and 50% of full sunlight. Both species showed similar morphological and physiological responses to exposure to different intensities of light. Although the total plant mass decreased under lower light conditions, the total leaf area per plant increased. The reason being that the leaf mass per plant did not decrease, while the leaf mass per unit area decreased. Leaf nitrogen and chlorophyll concentrations and the chlorophyll/nitrogen ratio increased under lower light conditions, whereas the chlorophyll a/b ratio decreased. These morphological and physiological changes may be seen as ways to increase light acquisition under low light conditions. Leaf atropine and scopolamine concentrations did not differ among the three light conditions for both species. In conclusion, both Datura species underwent morphological and physiological changes under low light conditions, enabling them to use carbon and nitrogen to increase light acquisition while maintaining their chemical defense capability.

Influences of small-scale oscillations on growth inhibition and ultrastructural changes of Microcystis cells.

We investigated the effects of small-scale oscillation (SSO) on toxic Microcystis cells. The oscillating device was made of silicon with two axes that had a diameter of ∼40 mm, and a frequency of 2.5 Hz was observed at 150 rpm. The SSO was effective in inhibiting Microcystis growth. Microcystin release was not observed, whereas cell density barely increased in the oscillating group. Cell size and morphology of the oscillating group were no different from the control group. However, cell quotas of chl.a and microcystin in the oscillating group were half the level of the control group. Crucially, a number of large-sized holes were observed and layered long linear thylakoids were rarely observed in the oscillating group. Therefore, SSO was found to be very effective in Microcystis growth inhibition, and it caused ultrastructural changes without damage to the cell membrane and subsequent microcystin release.

Design and daytime performance of laser-induced fluorescence spectrum lidar for simultaneous detection of multiple components, dissolved organic matter, phycocyanin, and chlorophyll in river water.

In this work, we developed mobile laser-induced fluorescence spectrum (LIFS) lidar based on preliminary experiments on the excitation emission matrix of a water sample and a method for reducing solar background light using the synchronous detection technique. The combination of a UV short-pulse laser (355 nm, 6 ns) for fluorescence excitation with a 10-100 ns short-time synchronous detection using a gated image-intensified multi-channel CCD of the fluorescence made the LIFS lidar operation possible even in daytime. The LIFS lidar with this construction demonstrated the potential of natural river/lake water quality monitoring at the Tenryu River/Lake Suwa. Three main components in the fluorescence data of the water, dissolved organic matter, phycocyanin, and chlorophyll, were extracted by spectral analysis using the standard spectral functions of these components. Their concentrations were estimated by adapting experimentally calibrated data. Results of long-term field observations using our LIFS lidar from 2010 to 2012 show the necessity of simultaneous multi-component detection to understand the natural water environment.

Microcystin release and Microcystis cell damage mechanism by alum treatment with long-term and large dose as in-lake treatment.

Most of our previous studies reported aluminum causes no cell damage or lysis, and no subsequent toxin release in conventional treatment of drinking water or in the laboratory, on the contrary, we investigated the effect of long-term and large-dose alum treatment, because the environmental conditions in lakes and treatment plants are widely different. The microcosm experiments were designed to simulate the effect of adding alum under the similar conditions of common lakes and reservoirs, and the bottle experiments were conducted to examine the budget or dynamics of microcystin after adding alum. In precipitate analyses, we also confirm the release and dynamics of microcystin and the damage mechanisms of Microcystis cells under alum treatment. In microcosms treated with alum alone, the extracellular microcystin-LR (MC-LR) concentration increased to approximately 82% in 7 days. Similar results were obtained in bottle experiments. By plotting the concentration of released microcystin over time, we inferred that the extracellular MC-LR concentration exponentially rose toward an asymptotic maximum. Moreover, in scanning electron microscope images, some cells exhibited torn membranes with miniscule traces of aluminum hydroxide coating. We conclude that alum treatment, particularly at maximum dosage administered over long periods, seriously damages Microcystis cells and induces microcystin release. Therefore, long-term application of large alum doses is not recommended as an in-lake treatment.

The removal of Microcystis ichthyoblabe cells and its hepatotoxin microcystin-LR during electrooxidation process using Pt/Ti electrodes.

Electrooxidation is widely used to remove harmful organic and inorganic substances as well as pathogenic microorganisms. This study investigates the removal of Microcystis ichthyoblabe cells and their hepatotoxin microcystin-LR by the electrooxidation process using Pt/Ti electrodes. Additionally, the morphology changes and cell sizes were determined by scanning electron microscopy and a particle size analyzer, respectively. The algal cells were severely damaged by the electrooxidation process. During the initial treatment, intracellular microcystin-LR was released from the cells, increasing the extracellular microcystin-LR concentration. The electrooxidation charge required to remove cells and MC-LR was 3 × 10(4) C and 6 × 10(4) C, respectively. The removal efficiencies of M. ichthyoblabe cells and microcystin-LR were insensitive to initial cell density, initial microcystin-LR concentration and solution conductivity, but were heavily reduced at large algal suspension volume. Therefore, to achieve simultaneous removal of Microcystis cells and their MC, it is necessary to control the volume of algal suspension.

Development of a UV laser-induced fluorescence lidar for monitoring blue-green algae in Lake Suwa.

We developed a UV (355 nm) laser-induced fluorescence (LIF) lidar for monitoring the real-time status of blue-green algae. Since the fluorescence spectrum of blue-green algae excited by 355 nm showed the specific fluorescence at 650 nm, the lidar was designed to be able to detect the 650 nm fluorescence as a surveillance method for the algae. The usefulness was confirmed by observation at Lake Suwa over four years (2005-2008). The detection limit of the LIF lidar was 16.65 mg/L for the blue-green algae, which is the range of concentrations in the safe level set by the World Health Organization.

The effect of alum coagulation for in-lake treatment of toxic Microcystis and other cyanobacteria related organisms in microcosm experiments.

Microcosm and bottle experiments were conducted to evaluate the effects of alum treatment on cyanobacteria-lysing organisms and microcystin-degrading bacteria as well as Microcystis cells, and to provide detailed evidence of Microcystis cell damage by investigating precipitated Microcystis cells. The alum concentration to be pH 6.0 is the maximum which does not cause toxicity by monomeric Al, therefore, this concentration was defined as maximum dose. Microcystis cells were considerably damaged by the alum treatment with maximum dose and long contact time. Seven days post-treatment, intracellular microcystin-LR was released into the extracellular environment in excess of 95 percent and chlorophyll a is not easily released from inside the cell, chl.a of precipitated Microcystis cells was also decreased to approximately 50 percent. Moreover, alum treatment caused damage to cyanobacteria-lysing organisms and microcystin-degrading bacteria, as well as to Microcystis cells. Therefore, it could be concluded that alum treatment with maximum dose (48 mg L(-1) as AI) is not suitable for removing cyanobacterial bloom without the release of cyanotoxin in reservoirs and ponds.

Bacterial and fungal community composition and community-level physiological profiles in forest soils.

We characterized the potential functioning and composition of the bacterial and fungal communities in the O and A horizons of forest soils using community-level physiological profile (CLPP) based on BIOLOG analysis, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S and 18S rDNA fragments, respectively. In addition, relationships between the potential functioning and the community composition in each horizon, and between the O and A horizons, were assessed using Procrustes analysis. For the bacterial and fungal communities, the CLPP and DGGE profile were clearly separated between the O and A horizons in a principal coordinate analysis except for the fungal CLPP. No significant links for CLPP and DGGE profile between the O and A horizons were observed for either bacterial or fungal communities, suggesting that different factors had considerable influence on the microbial communities between the O and A horizons. Significant couplings between bacterial and fungal DGGE profiles (p <0.05 for O horizon; p <0.01 for A horizon), and between bacterial and fungal CLPPs (p = 0.001 for O horizon; p <0.01 for A horizon), were observed in the O and A horizons, implying that common factors strongly influenced the bacterial and fungal communities in each horizon. Although a significant correlation was observed between bacterial community composition and the potential functioning in the A horizon (p <0.01), such a correlation was not observed for the fungal community in the A horizon, and for the bacterial and fungal communities in the O horizon. This finding suggested that potential functioning, which would reflect only rapidly growing microorganisms, was not strongly associated with the composition of the entire microbial community. Further studies are needed to unravel the factors shaping the composition and functioning of microbial communities in forest soils.

Ecological niche separation in the Polynucleobacter subclusters linked to quality of dissolved organic matter: a demonstration using a high sensitivity cultivation-based approach.

The free-living, cosmopolitan, freshwater betaproteobacterial bacterioplankton genus Polynucleobacter was detected in different years in 11 lakes of varying types and a river using the size-exclusion assay method (SEAM). Of the 350 strains isolated, 228 (65.1%) were affiliated with the Polynucleobacter subclusters PnecC (30.0%) and PnecD (35.1%). Significant positive correlations between fluorescence in situ hybridization and SEAM data were observed in the relative abundance of PnecC and PnecD bacteria to Polynucleobacter communities (PnecC + PnecD). Isolates were mainly PnecC bacteria in the samples with a high specific UV absorbance at 254 nm (SUVA(254) ), and a low total hydrolysable neutral carbohydrate and amino acid (THneutralCH + THAA) content of the dissolved organic matter (DOM) fraction, which is known to be correlated with a high humic content. In contrast, the PnecD bacteria were abundant in samples with high chlorophyll a and/or THneutralCH + THAA concentrations, indicative of primary productivity. With few exceptions, differences in the relative abundance of PnecC and PnecD in each sample, determined using a high-sensitivity cultivation-based approach, were due to DOM quality. These results suggest that the major DOM component in the field, which is allochthonously or autochthonously derived, is a key factor for ecological niche separation between PnecC and PnecD subclusters.

Dissolved organic nitrogen, dinoflagellates and cyanobacteria in two eutrophic lakes: Analysis by inferential modelling.

The Lake Suwa (Japan) has a history of non-N-fixing Microcystis blooms. Lake Kinneret (Israel) experienced multiannual periods of sole domination by the dinoflagellate Peridinium gatunense and periods dominated seasonally by P. gatunense or cyanobacteria. Extensive studies have been carried out in both lakes regarding the role of dissolved inorganic nitrogen and phosphorus as drivers of primary productivity. There is growing evidence that dissolved organic nitrogen (DON) compounds also influence not only biomass and structure of phytoplankton communities but also microcystin production. This study focuses on relationships of DON with: (1) population dynamics of Microcystis spp. and concentrations of microcystins in Lake Suwa, and (2) population dynamics of P. gatunense as well as N- and non-N-fixing cyanobacteria in Lake Kinneret. Modelling results for historical data of Lake Suwa by means of the hybrid evolutionary algorithm HEA revealed that the prediction of abundances of four Microcystis species and concentrations of cyanotoxins achieved higher coefficients of correlation when DON/DIN-ratios were included as drivers. Population dynamics of P. gatunense in Lake Kinneret appeared to have a strong inverse relationships with DON/DIN-ratios reflected by inferential models of HEA with higher coefficients of correlation when driven by DON/DIN-ratios. When DON/DIN-ratios were included as drivers, models of Microcystis spp. in Lake Kinneret performed higher coefficients of determination compared to models of N-fixing cyanobacteria. The study highlights the need to consider DON for improved understanding and management of population dynamics of cyanobacteria and dinoflagellates in freshwater lakes.

Complete Genome Sequence of a Microcystin-Degrading Bacterium, Sphingosinicella microcystinivorans Strain B-9.

Sphingosinicella microcystinivorans strain B-9 has the ability to degrade cyanobacterial hepatotoxic cyclic peptides, microcystins, and nodularins. This is the first report of the complete genome sequence of the microcystin-degrading bacterium.

Elucidation and short-term forecasting of microcystin concentrations in Lake Suwa (Japan) by means of artificial neural networks and evolutionary algorithms.

Non-supervised artificial neural networks (ANN) and hybrid evolutionary algorithms (EA) were applied to analyse and model 12 years of limnological time-series data of the shallow hypertrophic Lake Suwa in Japan. The results have improved understanding of relationships between changing microcystin concentrations, Microcystis species abundances and annual rainfall intensity. The data analysis by non-supervised ANN revealed that total Microcystis abundance and extra-cellular microcystin concentrations in typical dry years are much higher than those in typical wet years. It also showed that high microcystin concentrations in dry years coincided with the dominance of the toxic Microcystis viridis whilst in typical wet years non-toxic Microcystis ichthyoblabe were dominant. Hybrid EA were used to discover rule sets to explain and forecast the occurrence of high microcystin concentrations in relation to water quality and climate conditions. The results facilitated early warning by 3-days-ahead forecasting of microcystin concentrations based on limnological and meteorological input data, achieving an r(2)=0.74 for testing.

Widespread dispersal and bio-accumulation of toxic microcystins in benthic marine ecosystems.

Freshwater cyanobacteria produce toxic microcystins (MCs), which travel from freshwater areas into the sea. The MCs produced by cyanobacteria in a freshwater reservoir were discharged frequently into the adjacent Isahaya Bay, remained in the surface sediments, and then accumulated in various macrobenthic animals on the seafloor. The MCs were transported further outside of Isahaya Bay (Ariake Bay), and the median values of the MC contents in the sediments were in the same levels in both bays, while their temporal variations were also similar during the study period. Therefore, the fluctuations of the MC contents in the surface sediments were physically controlled by the timing of the discharge from the reservoir. The MC contents in polychaetes and oysters collected in Isahaya Bay increased markedly during winter. The median values of the carbon-based MC contents in the sediments, primary consumers, and secondary consumers in the bay were 87, 160, and 250 ngMC gC-1, respectively. These results demonstrated bio-accumulation at lower trophic levels in benthic marine ecosystems. An understanding of the processes occurring between sediments and macrobenthic animals is important for clarifying MC dynamics in ecosystems.

Design of a portable urine glucose monitoring system for health care.

This paper describes the design of a monitoring system that can be used to measure urine glucose during daily life. It consists of a bio-chemical sensor, hardware with PIC microcontroller and control circuits, and signal analyzing part. To evaluate the performance, we compared the analyzed glucose levels of the developed system to a standard instrument, YSI glucose analyzer, based on regression analysis using standard glucose solutions mixed with urine. Also, standard deviation and coefficient of variation were computed. In conclusion, the developed system showed it could be used for the measurement of urine glucose.

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells.

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293-OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293-OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity.

Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes.

mlrA is the only microcystin-degrading gene detected in Sphingomonas sp. MJ-PV. The gene has an extremely rare nucleotide sequence and homologous genes have not yet been discovered in the DNA database. We discovered the existence of a gene homologous to mlrA in new microcystin-degrading bacteria, MD-1 and Y2. These strains possessed mlrA homologues, and the identities of the genes of MD-1 and Y2 with the corresponding MJ-PV exceeded 98% and 84%, respectively. On the other hand, the mlrA gene was not detected in laboratory strains of the closely related Sphingomonas spp. strains employing hemi-nested polymerase chain reaction detection using two primer sets. Although the microcystin-degrading bacteria were closely related strains, they did not cluster together as the same species. We can conclude that the mlrA gene is conserved in three different bacterial species, and it is unique to microcystin degraders but not to the genus Sphingomonas.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment.

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 microg g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 microg g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 microg g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood>liver>muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters.

Inhibitory effect of naringin on microcystin-LR uptake in the freshwater snail Sinotaia histrica.

Gastropods are an important food source for aquatic animals, and have been demonstrated to transfer microcystin (MC) to higher trophic levels through the food web. In this study, we performed an oral administration experiment to evaluate whether naringin can inhibit MC-LR uptake in the freshwater snail Sinotaia histrica. We also observed the effect of MC-LR on the organizational pathology of the hepatopancreas in S. histrica. Following exposure to cells of Microcystis ichthyoblabe, S. histrica showed vacuolization and separation of the basal lamina from cells in the hepatopancreas. Initial treatment with 1mM naringin resulted in the prevention of MC-LR uptake rate by approximately 60% over 8days, whereas initial treatment with 10mM naringin suppressed microcystin uptake in 2days, despite an increase in MC-LR levels in the snail from days 5 to 8. With continuous treatment of 10mM naringin, the uptake prevention rate was 100%. Overall, we observed a strong inhibitory effect against MC-LR with naringin treatment. This study provides a potential mechanism to prevent the uptake of microcystin in the aquatic food web, thereby limiting its toxicity in cyanobacterial bloom-polluted areas where the environment can be controlled and may have further applications in the aquaculture of gastropods.

Detoxification of microcystin-LR in water by Portulaca oleracea cv.

Microcystin-LR (0.02 µg/ml) in the hydroculture medium of Portulaca oleracea cv., became below the detection level (<0.0001 µg/ml) by HPLC analysis after 7 days. The toxicity of microcystin estimated with protein phosphatase inhibition assay, however, remained at 37% of the initial level, indicating that microcystin-LR was transformed by P. oleracea cv. into unknown compound(s) of lower toxicity.

Synthesis of iron composites on nano-pore substrates: identification and its application to removal of cyanide.

Two types of nano-pore substrates, waste-reclaimed (WR) and soil mineral (SM) with the relatively low density, were modified by the reaction with irons (i.e. Fe(II):Fe(III)=1:2) and the applicability of the modified substrates (i.e. Fe-WR and Fe-SM) on cyanide removal was investigated. Modification (i.e. Fe immobilization on substrate) decreased the BET surface area and PZC of the original substrates while it increased the pore diameter and the cation exchange capacity (CEC) of them. XRD analysis identified that maghemite (γ-Fe(2)O(3)) and iron silicate composite ((Mg, Fe)SiO(3)) existed on Fe-WR, while clinoferrosilite (FeSiO(3)) was identified on Fe-SM. Cyanide adsorption showed that WR adsorbed cyanide more favorably than SM. The adsorption ability of both original substrates was enhanced by the modification, which increased the negative charges of the surfaces. Without the pH adjustment, cyanide was removed as much as 97% by the only application of Fe-WR, but the undesirable transfer to hydrogen cyanide was possible because the pH was dropped to around 7.5. With a constant pH of 12, only 54% of cyanide was adsorbed on Fe-WR. On the other hand, the pH was kept as 12 without adjustment in Fe-WR/H(2)O(2) system and cyanide was effectively removed by not only adsorption but also the catalytic oxidation. The observed first-order rate constant (k(obs)) for cyanide removal were 0.49 (± 0.081) h(-1). Moreover, the more cyanate production with the modified substrates indicated the iron composites, especially maghemite, on substrates had the catalytic property to increase the reactivity of H(2)O(2).