mcyD Gene Research Articles

Response of cytotoxin production ability to gene expression and cell molecular structure of Microcystis aeruginosa FACHB-905.

To investigate the inhibitory mechanism of artemisinin sustained-release microspheres (ASMs) on Microcystis aeruginosa (M. aeruginosa) from the molecular level, prx, psbA, fabZ, and mcyD were studied, and the cell death mode were also explored. The results showed that expression of prx was slightly up-regulated, while the expression of psbA, fabZ, and mcyD was significantly reduced. It can infer that oxidant damage and photic damage are the main mechanisms for the algicidal effect of ASMs on M. aeruginosa. It can be seen from the changes in cell morphology and structure that microspheres stress triggers apoptosis-like cell death, and the cell membrane is intact effectively preventing the leakage of microcystin-LR (MC-LR). Moreover, the down-regulation of mcyD gene also played major role in less extracellular MC-LR than intracellular MC-LR. It was concluded that the ASMs will not cause secondary ecological hazards while killing algae cells and have good application prospects.

A Novel Microcystin-Producing Cyanobacterial Species from the Genus Desmonostoc, Desmonostoc alborizicum sp. nov., Isolated from a Water Supply System of Iran

A qanat or kariz is a slightly sloping underground aqueduct used to transport water from wells or aquifers to the surface for irrigation and drinking supply. A cyanobacterial strain was isolated from a cyanobacterial mat colonizing the wall of a qanat in Golestan province, Gorgan City, Iran. Fragments of 16S rRNA, mcyG, and mcyD genes were amplified and sequenced, as well as the 16S-23S internal transcribed spacer (ITS). After microscopic examination, the isolate was related to a morphotype of Nostoc sensu lato group, with similar characteristics to Desmonostoc. The 16S rRNA phylogenetic analysis placed the isolate into the typical cluster of the recently proposed genus Desmonostoc. Morphological analysis revealed distinctive characteristic and secondary 16S-23S rRNA structures derived from comparative analysis, which did not match known species of Desmonostoc. These results lead us to propose a novel Desmonostoc species, Desmonostoc alborizicum, which was described and compared with similar taxa. Furthermore, for the first time a potentially toxic species of Desmonostoc was isolated from a water supply, since the mcyD and mcyG genes of the microcystin synthetase (mcy) cluster were successfully sequenced. Using mass spectrometry, detectable amounts of the hepatotoxin microcystin-LR and -RR, along with demethylated variants, were present in cell extracts of the Desmonostoc strain. Our findings contribute to a deeper understanding of the diversity, systematics, and occurrence of the genus Desmonostoc.

Evaluating the effectiveness of various biochemical and molecular techniques to assess microcystin risk during the onset process of Microcystis blooms (delay-development stages)

A variety of cyanotoxins (e.g., microcystin) threaten water safety globally, and biochemical methods and molecular techniques have been used to assess microcystin risk. When a toxic bloom breaks out and associated microcystin risk will be changed, but whether these techniques is proper to assess microcystin risk for this process, remains unknown. Herein, the onset process (delay-development stages) of a bloom was simulated using a toxic strain of Microcystis aeruginosa-915, and the microcystin risk was assessed by biochemical techniques (ELISA and HPLC) and molecular techniques (DNA- and RNA-based qPCR). Results showed that indirect ELISA and HPLC could certify microcystin risk via accurate quantification of microcystin concentrations. Compared to indirect ELISA, direct ELISA underestimated microcystin risk due to a decrease of microcystin quantifications (20–55%) resulting from the competitive inhibition by elevated AOMs at development stage. Reliability of mcyD gene copies using DNA-based qPCR to indirectly assess microcystin risk was affected by microcystin-producing biomass at development stage. RNA-based qPCR could be a promising tool to achieve early-warning and real-time assessments of microcystin risk for this process. Besides, the ratio of mRNA/DNA copies of mcyD were proposed as an indicator, since it performed better (r = 0.93, p < 0.01) to indirectly assess microcystin risk than their total copies (r = 0.86, p < 0.01) for bloom samples. These results would provide a detailed guideline for water managers to employ proper techniques for assessments of microcystin risk during the onset process of a toxic bloom.

Oxidation to Control Cyanobacteria and Cyanotoxins in Drinking Water Treatment Plants: Challenges at the Laboratory and Full-Scale Plants

The impact of oxidation on mitigation of cyanobacteria and cyanotoxins in drinking water treatment sludge was investigated at the laboratory and treatment plant scales. Two common oxidants, KMnO4 (5 and 10 mg/L) and H2O2 (10 and 20 mg/L) were applied under controlled steady-state conditions. Non-oxidized and oxidized sludge was left to stagnate in the dark for 7 to 38 days. Controlled laboratory trials show that KMnO4 and H2O2 decreased cell counts up to 62% and 77%, respectively. The maximum total MC level reduction achieved after oxidation was 41% and 98% using 20 mg/L H2O2 and 10 mg/L KMnO4, respectively. Stagnation caused cell growth up to 2.6-fold in 8 out of 22 oxidized samples. Microcystin (MC) producer orders as Chroococcales and Synechococcales were persistent while Nostocales was sensitive to combined oxidation and stagnation stresses. In parallel, two on-site shock oxidation treatments were performed in the DWTP’s sludge holding tank using 10 mg/L KMnO4. On-site shock oxidation decreased taxonomic cell counts by up to 43% within 24 h. Stagnation preceded by on-site shock oxidation could increase total cell counts by up to 55% as compared to oxidation alone. The increase of cell counts and mcyD gene copy numbers during stagnation revealed the impact of oxidation/stagnation on cyanobacterial cell growth. These findings show the limitations of sludge oxidation as a strategy to manage cyanobacteria and cyanotoxins in sludge and suggest that alternative approaches to prevent the accumulation and mitigation of cyanobacteria in sludge should be considered.

Metagenomic study to evaluate functional capacity of a cyanobacterial bloom during oxidation

Pre-oxidation can be used against cyanobacteria at the water treatment plant intake to improve cell removal efficiency in down flow processes and reduce cyanotoxins concentrations. In this study, shotgun metagenomic sequencing was used to describe the functional capacity of a cyanobacterial bloom (at Lake Champlain, southern Quebec, Canada) before and after pre-oxidation using Cl2, KMnO4 and H2O2. The bloom samples were associated with two functional profile assemblages: that of August 1st (onset of the bloom) characterized by enrichment of genes related to nutrient uptake and that of August 13th-29th (towards the end of the sampling) associated with competition for resources and repair such as Photosynthesis, Protein metabolism and DNA metabolism. Different functional profile responses to oxidation with Cl2, KMnO4 and H2O2 was also identified as two-time points during the bloom (at the August 1st, and August 29th). On August 1st, chlorinated samples showed a progressive shift in functional profile: starting by acquiring and sequestering nutrient sources (e.g. Iron acquisition, carbohydrates) at low chlorine exposure (CT, concentration X contact time) level, followed by showing a stronger tendency toward dormancy and sporulation genes at high CT. Our results showed that following high CT of H2O2, the relative abundance of the cyanobacterial biomarkers decreased, regardless of the dominant cyanobacterial genus. The toxicity of the bloom before and after oxidation samples was assessed by droplet digital PCR (ddPCR) to measure the mcyD gene. Our results showed significant positive correlation between the mcyD gene copies number and microcystin concentrations in the bloom samples (before the oxidation). However, such correlation was not observed after oxidation. These results suggest that ddPCR can only be used to evaluate bloom toxicity before oxidation.

Using quantitative real-time polymerase chain reaction (qRT-PCR) for detection microcystin producing cyanobacteria

Introduction: Cyanobacterial blooms (CBs) have become a growing concern worldwide. In the natural environment, potentially toxic (can produce toxins) and non-toxic (can not produce toxins) colonies often co-exist within a bloom.&#x0D; Methods: The present study aimed to quantify toxic and non-toxic cells of cyanobacteria in the Tri An Reservoir (TAR) using a quantitative real-time polymerase chain reaction (qRT-PCR).&#x0D; Results: Results showed that the Microcystis genus dominated the cyanobacterial communities in the TAR. Microcystis was also the primary microcystins (MC) producing cyanobacteria in the water. Total cyanobacteria and Microcystis cells ranged from 152103 to 27106 copy/L and from 105103 to 19106 copy/L, respectively. The cell number of potentially MC-producing cyanobacteria (corresponding to the Microcystis mcyD gene) varied from 27103 to 13106 copy/L. MC concentrations often present in raw water with a concentration of up to 4.8 mg/L. Our results showed that the MC concentration in raw water was positively correlated with the mcyD copy number, suggesting that Microcystis spp. are the main toxin producers in the TAR's surface water.&#x0D; Conclusion: Our study suggested that qRT-PCR techniques and traditional count are comparable and could be used to quantify cyanobacteria. In addition, the qRT-PCR techniques can determine the toxic cyanobacterial cells and could be used as a tool for early monitoring of toxic cyanobacteria in lakes and reservoirs.

Hormones induce the metabolic growth and cytotoxin production of Microcystis aeruginosa under terpinolene stress

Several organic compounds released into the aquatic environment have a detrimental impact on humans and other organisms. There is a lack of knowledge about natural hormones and herbicides on non-target organisms, including cyanobacteria. In this study, the response of Microcystis aeruginosa to four phytohormones, indole-3-acetic acid (IAA; 10−5), zeatin (ZT; 10−5), abscisic acid (ABA; 10−7), and brassinolide (BRL; 10−9 mol/L), exposed to terpinolene (TPN; (0.44, 0.88, 1.17, or 1.62 mmol/L) at the cellular and genetic levels were investigated. The results showed that TPN could inhibit the growth and photosynthetic activities and stimulate microcystins (MCs) of M. aeruginosa at various levels through the co-occurrence of oxidative stress, antioxidant defense activities, and an imbalance of the antioxidative system. Hormones played critical roles in the growth promotion and photosynthetic activity by enhancing the antioxidant defense mechanisms and MCs production of M. aeruginosa under TPN stress in both hormone and TPN dose-dependent manner. The growth performance and photosynthetic activities of M. aeruginosa were significant with IAA (p < 0.01) and BSL (p < 0.05) compared to ZT and ABA, as TPN concentrations increased. Hormones stimulated the MCs production significantly BSL (p < 0.05) at various levels and protected the cells against TPN-induced oxidative stress and expression of mcyB and mcyD genes involve in MCs synthesis. Our results indicated that hormone contamination in eutrophic lakes might increase the risk of Microcystis aeruginosa bloom and microcystin production with the TPN association.

FurA-Dependent Microcystin Synthesis under Copper Stress in Microcystis aeruginosa.

Massive blooms of cyanobacteria frequently occur with microcystin (MC) production. Cyanobacteria are exposed to copper stresses such as copper algaecides which are often used to remove cyanobacterial blooms. However, copper increased the MC production of cyanobacteria, and the underlying mechanism remains unclear. The present study investigated the relationship between copper exposure (0.5 and 3 µM) and MC synthesis in Microcystis aeruginosa PCC 7806. The study concluded that the content of intracellular MCs increased by nearly two times both in 0.5 and 3 µM copper. High-throughput RNA sequencing (RNA-seq) provided evidence that copper mainly attacked Fe–S clusters, with evidence of changes in iron, sulfur, iron uptake regulators (fur), glutaredoxins and dehydratase genes. The transcription of numbers of genes implicated in iron uptake, MC synthesis and furA was also evaluated with quantitative real-time PCR (qRT-PCR). In these three Cu treatment groups, the amount of MCs increased as copper elevated. As the expression of mcyD gene was directly regulated by FurA and copper ions affected the expression of the FurA-related genes, we believed that MC synthesis genes were controlled by copper. This study has made a further understanding of the mechanism of the increase in MC synthesis of M. aeruginosa PCC 7806 treated with copper-based algaecides. We aimed to understand the mechanism of copper ion influencing the synthesis of MCs.

A CMOS Read-Out IC for Cyanobacteria Detection With 40 nApp Sensitivity and 45-dB Dynamic Range

This paper presents a low-power high-sensitivity CMOS read-out integrated circuit (ROIC) realized in a 0.13 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$ {\mu }\text{m}$ </tex-math></inline-formula> CMOS technology for cyanobacteria detection and monitoring with the current sensing capability of nano-ampere levels. The proposed CMOS ROIC consists of a current-conveyor transimpedance amplifier (CC-TIA) and a 9 bit successive approximation register (SAR) analog-to-digital converter (ADC). Electrically measured results of the proposed ROIC demonstrate 98.6 dB <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\Omega $ </tex-math></inline-formula> transimpedance gain, 1.19 pA/ <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\surd $ </tex-math></inline-formula> Hz noise current spectral density, and 1.8 mA current consumption from a single 1.2 V supply. The ROIC can precisely recover the digital output codes corresponding to the input current levels of 40 nA <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">pp</sub> ~ 7.2 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu$ </tex-math></inline-formula> A <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">pp</sub> (i.e., 45 dB dynamic range). Optical experiments utilizing a 405 nm wavelength laser source and a low-cost PIN-photodiode confirm the fluorescence intensity with respect to the quantity of cyanobacteria, demonstrating that the normalized voltage (QD <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">655</sub> /QD <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">565</sub> ) is a linear function of the mcyD gene copy numbers. The CMOS ROIC chip occupies the area of 1.1 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\times$ </tex-math></inline-formula> 2.0 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> .

Short Term Effects of Hurricane Irma and Cyanobacterial Blooms on Ammonium Cycling Along a Freshwater–Estuarine Continuum in South Florida

Lacustrine and coastal systems are vulnerable to the increasing number and intensity of tropical storms driven by climate change. Strong winds associated with tropical storms can mobilize nutrients in sediments and alter nitrogen and phosphorus cycling, leading to amplification of preexisting conditions, such as eutrophication and cyanobacterial blooms (cyanoHABs). In 2016, Florida declared a State of Emergency within and downstream of Lake Okeechobee (LO) due to toxic cyanobacterial blooms (primarily Microcystis). The blooms originated in LO, but flood control measures released water from LO to the brackish St. Lucie Estuary (SLE). In September 2017, Hurricane Irma traversed the Florida peninsula with sustained winds exceeding 160 km h-1, generating torrential rains over the watershed. We quantified ammonium (NH4+) regeneration and potential uptake rates, and Microcystis toxin gene (mcyD) abundance in LO and SLE during the massive bloom in July 2016, the bloom in August 2017 (two weeks before Irma), and 10 days after Hurricane Irma landfall. In 2016, cyanoHABs were present in both LO and SLE, and potential NH4+ uptake rates were high in both systems. In 2017, the bloom was constrained to LO, and potential NH4+ uptake rates in LO exceeded those in SLE, and mcyD gene abundance was greater in LO than SLE. Post Hurricane Irma, potential NH4+ uptake rates decreased significantly in LO and SLE, while mcyD gene abundance decreased in LO and increased slightly in SLE. Average NH4+ regeneration rates could support 25–40% of water column potential NH4+ demand in the lake and, when extrapolated to the entire LO water column, exceeded external nitrogen loading. These results emphasize the importance of internal NH4+ recycling for bloom expansion and toxicity in the lake and downstream estuaries. In 2018, the cyanobacterial bloom in the Okeechobee region was one of the largest recorded and is presumed to be driven by the aftermath of Hurricane Irma. Large-scale blooms have also been observed in SLE, likely due to LO flushing and decreased salinity post hurricane. Thus, results from this study support predictions that increased frequency and strength of tropical storms will lead to more intense blooms in aquatic systems.

Contrasting histories of microcystin-producing cyanobacteria in two temperate lakes as inferred from quantitative sediment DNA analyses

Pilon S, Zastepa A, Taranu ZE, Gregory-Eaves I, Racine M, Blais JM, Poulain AJ, Pick FR. 2018. Contrasting histories of microcystin-producing cyanobacteria in two temperate lakes as inferred from quantitative sediment DNA analyses. Lake Reserv Manage. 35:102–117.The incidence and abundance of microcystin-producing cyanobacteria were compared between 2 temperate lakes over the past ∼150–175 yr by combining analyses of sediment DNA (qPCR of 4 target genes and high-throughput sequencing of a cyanobacterial 16S rRNA gene fragment) and sediment microcystin concentrations. In Lake of the Woods (LoW), toxic cyanobacteria were only detected beginning in the early 1980s based on gene copy numbers of the microcystin-synthesizing gene (mcyD), and by the early 2000s based on sediment microcystins; both these proxies for toxic cyanobacteria were significantly correlated. In contrast, in Baptiste Lake (Alberta), both mcyD gene copy numbers and microcystins were detected as far back as ∼1830, before European settlement of the region. Total sediment microcystins in this lake were not correlated with mcyD gene copy numbers, but the latter correlated with 2 other cyanobacterial genes targeted. These results point to a relatively recent emergence and increasing dominance of microcystin-producing cyanobacteria in LoW that coincides with the documented rise in regional temperatures rather than changes in nutrient loading. In contrast, toxic blooms are not a new phenomenon in Baptiste Lake, as microcystin-producing cyanobacteria were clearly part of the ecosystem prior to anthropogenic perturbations; this lake has also experienced a recent increase in microcystins coincident with further nutrient enrichment. High-throughput sequencing of the cyanobacterial 16S rRNA gene indicated that bloom-forming and potentially toxigenic Dolichospermum (including related Nostocales) and Microcystis were present but the former has been the dominant genus over the past 175 yr.

Diversity of microcystins and non-microcystin-producing &lt;i&gt; Microcystics &lt;/i&gt; population in the Dau Tieng reservoir, Vietnam

In this study, a 16S rDNA clone library was used to evaluate the diversity of potential microcystin producing cyanobacteria and polymerase chain reaction (PCR) was used to analyze the diversity of this population based on the presence of the mcyD gene in samples collected during a bloom season. The results revealed that Microcystis genera from the Dau Tieng reservoir (DTR) contained several genotypes. The isolated Microcystis strains from the DTR were found to consist both toxic and non-toxic species within species and population. In total 35 strains acounted for 58% were found to produce a mixture of microcystins (MCs). All samples of cyanobacterial blooms in the DTR contained high amount of MCs which could pose health risk to aquatic systems and local people.

DETECTION OF POTENTIALLY TOXIGENIC MICROCYSTIS STRAINS FROM DAU TIENG RESERVOIR

Microcystis is a bloom-forming, common cyanobacterium in Dau Tieng reservoir used for public water supply. To assess the presence of potentially microcystin-producing Microcystis, molecular techniques were conducted and acute toxicity bioassays were performed with the microcrustacean Daphnia magna exposed to cyanobacterial crude extracts. Potentially toxigenic of isolated strains was characterized by amplifying mcyD genes and identification of Microcystis was confirmed by 16S rRNA amplification. Microcystins (MCs) concentration in bloom samples and cultured strains were quantified by High Performance Liquid Chromatography (HPLC). Results showed that there were 9/15 strains showed positive with the mcyD marker indicating that they are toxic strains. Three MCs variants including MC-RR, -LR and -YR were found in all extracts of toxic strains with the highest concentration of 1,218 μg/g dry weight (DW). The acute toxicity bioassays revealed that both toxic and non-toxic crude extracts elicited significant lethal effects on the tested animal with LC50 values ranged from 189-411 mg DW/L. The toxic effects of isolated strains were independent from the MCs concentration in some strains suggesting the presence of other metabolites contributed to the biological effects. In conclusion, microcystin-producing Microcystis from the Dau Tieng reservoir warn about possible toxic effects for aquatic biota and human health.

Response of microcystin biosynthesis and its biosynthesis gene cluster transcription in Microcystis aeruginosa on electrochemical oxidation

ABSTRACTMicrocystin-LR (MC-LR), which is one of the most commonly found microcystins (MCs) in fresh water, has been proved to be a potential tumour promoter and classified as 2B by the International Agency for Research on Cancer. MC-LR decomposition and inhibition of MC-LR production in Microcystis aeruginosa were investigated under electrolysis condition using an electrolysis cell consisting of Ti/Pt electrodes and Nafion membrane. The relationship between the decrease in MC-LR concentration and transcription of MC-LR synthesis gene clusters was determined by performing real-time reverse transcription polymerase chain reaction (RT-qPCR) to monitor changes in the levels of transcription encoding mcyB and mcyD (cDNA to DNA) in M. aeruginosa NIES 1086 under electrolysis condition and three different conditions (i.e. oxygenated, air aerated and unaerated) as controls. Cell density decreased from day 2 under electrolysis than under the three controls. Intracellular MC-LR concentration was approximately 33 fg cell−1 under electrolysis from days 4 to 8, while those in the other conditions ranged in 40–50 fg cell−1. The mcyB transcription continuously decreased from day 2 to nondetectable level in day 6 under electrolysis, while this transcription was stabilised under the three controls. This result suggested that oxidative stress, such as hydroxyl radicals, played an important role in the down-regulation of mcyB and mcyD gene transcription level and the MC-LR concentration and cell density of M. aeruginosa.

Portable Cyanobacteria Detection Fluorometer with Current-conveyor Transimpedance Amplifiers

A portable fluorometer has been developed for cyanobacteria detection by exploiting commercially available low-cost photodiodes. Particularly, the front-end detection circuit was integrated on-chip by using a 0.11-μm CMOS process to realize the high transimpedance gain of 99.7 dBΩ with low current consumption of 1.4 mA from a single 1.5-V supply, hence providing a low-power solution with small form-factor in the applications of portable cyanobacteria detection. For measuring fluorescence intensity of cyanobacteria, two quantum dots of QDSUB565/SUB and QDSUB655/SUB were employed into a NanoGene assay for mcyD gene(synthetase of toxic microcystin) detection of Microcystis aeruginosa, resulting in two different readout voltages. Various operational parameters (e.g. distance, angle between laser emission and vial sample, etc) were investigated for the proposed fluorometer. Measurements demonstrated that the mcyD gene hybridized with QDSUB565/SUB and QDSUB655/SUB was quantitatively detected, i.e. the normalized output voltage (QDSUB655/SUB/QDSUB565/SUB) was linearly proportional to the mcyD gene copy numbers (y = 0.25 log x – 0.25). Besides, the wireless transfer of the quantitative data was performed via Bluetooth.

Detection and Quantification of Toxin-Producing Microcystis aeruginosa Strain in Water by NanoGene Assay.

We demonstrated the quantitative detection of a toxin-producing Microcystis aeruginosa (M. aeruginosa) strain with the laboratory protocol of the NanoGene assay. The NanoGene assay was selected because its laboratory protocol is in the process of being transplanted into a portable system. The mcyD gene of M. aeruginosa was targeted and, as expected, its corresponding fluorescence signal was linearly proportional to the mcyD gene copy number. The sensitivity of the NanoGene assay for this purpose was validated using both dsDNA mcyD gene amplicons and genomic DNAs (gDNA). The limit of detection was determined to be 38 mcyD gene copies per reaction and 9 algal cells/ml water. The specificity of the assay was also demonstrated by the addition of gDNA extracted from environmental algae into the hybridization reaction. Detection of M. aeruginosa was performed in the environmental samples with environmentally relevant sensitivity (~105 algal cells/ml) and specificity. As expected, M. aeruginosa were not detected in nonspecific environmental algal gDNA over the range of 2 × 100 to 2 × 107 algal cells/ml.

Portable Fluorometer for Cyanobacteria Detection

A portable system for fluorescence measurement has been developed for cyanobacteria detection. Exploiting commercially available low-cost photodiodes and a single 9 V supply, the system was able to measure two fluorescence emissions simultaneously. Output voltages resulting from the fluorescence intensity were measured by utilizing two quantum dots of QD565 and QD655. These quantum dots were employed into a NanoGene assay for mcyD gene (synthetase of toxic microcystin) detection of Microcystis aeruginosa. We investigated various operational parameters (e.g., distance, angle between laser emission and sample, and so on) for the proposed system. Measurements indicated the highest output voltage signal was obtained at a distance of 0.5 cm with the right angle (90°) between the laser source and a sample vial. Also, the mcyD gene hybridized with QD565 and QD655 was quantitatively detected by the proposed detection system, i.e., the normalized output voltage (QD655/QD565) was linearly proportional to the mcyD gene copy numbers (y = 0.41 log $x +6.6$ , $\text{r}^{2} =0.92$ ). Finally, the quantitative data were transferred wirelessly to the readout via Bluetooth.

Fecal contamination and Microcystis in drinking-water sources of rural Cambodia using PCR and culture-based methods

Rural communities within low-income countries frequently rely on a range of drinking-water sources, and each water source varies in its potential for biological contamination. The extent and source of biological contamination in primary drinking sources within Kien Svay, Kandal, Cambodia, were determined by fecal indicator bacteria (FIB) measurements, 16S rDNA genetic markers for human and bovine fecal Bacteroides, presence of the bloom-forming Microcystis species, and the microcystin toxin mcyD gene marker. Thirteen wells, 11 rain barrels, 10 surface-water sites, and five sediment samples were examined during the dry and wet seasons. Surface water was commonly contaminated with FIB, with up to 1.02 × 105Enterococcus sp., 6.13 × 104E. coli, and 2.91 × 104 total coliforms per 100 mL of water. Human and bovine Bacteroides were detected in 100 and 90% of the surface water samples, respectively. Concentrations of FIB in rain-barrels varied by site, however 91% contained human Bacteroides. Microcystis cells were found in 90% of surface water sites, with many also containing microcystin gene mcyD, representing the first report of microcystin-producing cyanobacteria in surface waters of Cambodia. The study results show that many potential drinking-water sources in Cambodia contain harmful bacterial and algal contaminants, and care should be taken when selecting and monitoring water options.

Distribution and Habitat Specificity of Potentially-Toxic Microcystis across Climate, Land, and Water Use Gradients.

Toxic cyanobacterial blooms are a growing threat to freshwater bodies worldwide. In order for a toxic bloom to occur, a population of cells with the genetic capacity to produce toxins must be present together with the appropriate environmental conditions. In this study, we investigated the distribution patterns and phylogeny of potentially-toxic Microcystis (indicated by the presence and/or phylogeny of the mcyD and mcyA genes). Samples were collected from the water column of almost 60 water bodies across widely differing gradients of environmental conditions and land use in Israel. Potentially, toxic populations were common but not ubiquitous, detected in ~65% of the studied sites. Local environmental factors, including phosphorus and ammonia concentrations and pH, as well as regional conditions such as the distance from built areas and nature reserves, were correlated with the distribution of the mcyD gene. A specific phylogenetic clade of Microcystis, defined using the sequence of the mcyA gene, was preferentially associated with aquaculture facilities but not irrigation reservoirs. Our results reveal important environmental, geospatial, and land use parameters affecting the geographic distribution of toxinogenic Microcystis, suggesting non-random dispersal of these globally abundant toxic cyanobacteria.

The combined effects of Dolichospermum flos-aquae, light, and temperature on microcystin production by Microcystis aeruginosa

The effects of light, temperature, and coculture on the intracellular microcystin-LR (MC-LR) quota of Microcystis aeruginosa were evaluated based on coculture experiments with nontoxic Dolichospermum (Anabaena) flos-aquae. The MC-LR quota and transcription of mcyB and mcyD genes encoding MC synthetases in M. aeruginosa were evaluated on the basis of cell counts, high-performance liquid chromatography, and reverse-transcription quantitative real-time PCR. The MC-LR quotas of M. aeruginosa in coculture with a 1/1 ratio of inoculum of the two species were significantly lower relative to monocultures 6-d after inoculation. Decreased MC-LR quotas under coculture conditions were enhanced by increasing the D. flos-aquae to M. aeruginosa ratio in the inoculum and by environmental factors, such as temperature and light intensity. Moreover, the transcriptional concentrations of mcyB and mcyD genes in M. aeruginosa were significantly inhibited by D. flos-aquae competition in coculture (P <0.01), lowered to 20% of initial concentrations within 8 days. These data suggested that coculture eff ects by D. flos-aquae not only reduced M. aeruginosa’s intracellular MC-LR quota via inhibition of genes encoding MC synthetases, but also that this eff ect was regulated by environmental factors, including temperature and light intensities.