Genomic architecture constrains macromolecular allocation in dinoflagellates.

Dinoflagellate genomes have a unique architecture that may constrain their physiological and biochemical responsiveness to environmental stressors. Here we quantified how nitrogen (N) starvation influenced macromolecular allocation and C:N:P of three photosynthetic marine dinoflagellates, representing different taxonomic classes and genome sizes. Dinoflagellates respond to nitrogen starvation by decreasing cellular nitrogen, protein and RNA content, but unlike many other eukaryotic phytoplankton examined RNA:protein is invariant. Additionally, 2 of the 3 species exhibit increases in cellular phosphorus and very little change in cellular carbon with N-starvation. As a consequence, N starvation induces moderate increases in C:N, but extreme decreases in N:P and C:P, relative to diatoms. Dinoflagellate DNA content relative to total C, N and P is much higher than similar sized diatoms, but similar to very small photosynthetic picoeukaryotes such as Ostreococcus. In aggregate these results indicate the accumulation of phosphate stores may be an important strategy employed by dinoflagellates to meet P requirements associated with the maintenance and replication of their large genomes.

Yellow clay modulates carbohydrate and glutathione responses in the harmful dinoflagellate Cochlodinium polykrikoides and leads to sedimentation.

The marine dinoflagellate Cochlodinium polykrikoides is a harmful algal bloom (HAB) species that severely impacts the environment and causes huge economic losses. Yellow clay (YC), considered to be a non-toxic and naturally-occurring material, represents an important step towards the direct control of HABs. In the present study, we evaluated the physiological and biochemical effects of YC on C. polykrikoides after exposures of up to 72 h. We observed little physiological changes in growth rate, chlorophyll a, lipid peroxidation, antioxidant enzymatic activities of superoxide dismutase and catalase, and activity of alkaline phosphatase after exposure to YC. Interestingly, YC significantly increased total carbohydrate and glutathione levels, affecting the physiology of the cells. These results indicate that total carbohydrate content may play an important role in cell-clay aggregation and it could be the main underlying mechanism that mitigates HAB cells via sedimentation.

Mass mortality of fish and water quality assessment in the tropical Adyar estuary, South India.

Mass mortality of fishes was reported at the Adyar estuary, South India, during November 2017. The probable reasons for fish mortality are analyzed in this paper. Critical assessments on water quality parameters including the metal concentrations, nutrients, and histology of gills and liver of fish (Mugil cephalus) isolated from the impact zone were performed. Among the metals observed, chromium showed levels (3.64 ± 0.001 mg L-1) much above the average permissible limits (0.1 mg L-1). The measured values of physico-chemical parameters in the impact zone are as follows: dissolved oxygen 4.7 ± 0.22 mg L-1, total alkalinity 132 ± 4 CaCO3 mg L-1, salinity 5.3 ± 0.3 PSU, temperature 27.8 ± 0.16 °C, nitrate, 1.66 ± 0.48 mg L-1, nitrite 0.01 ± 0.0008 mg L-1, ammonia 0.03 ± 0.001 mg L-1, phosphate 1.52 ± 0.002 mg L-1, and silicate 13.85 ± 3.1 mg L-1. The low salinity could have escalated the toxicity of the metal. In addition, histology of gills and liver showed cellular necrosis, epithelial lifting, hyperplasia, edema, mucous cell proliferation in the gills, cytoplasmic vacuolation of hepatocytes, and degeneration of liver which reveal that chromium toxicity is the most probable cause for mass mortality.

Photosynthetic and biochemical responses of the freshwater green algae Closterium ehrenbergii Meneghini (Conjugatophyceae) exposed to the metal coppers and its implication for toxicity testing.

The freshwater green algae Closterium is sensitive to water quality, and hence has been suggested as ideal organisms for toxicity testing. In the present study, we evaluated the photosynthetic and biochemical responses of C. ehrenbergii to the common contaminants, coppers. The 72 h median effective concentrations (EC50) of CuSO4 and CuCl2 on the test organism were calculated to be 0.202 mg/L and 0.245 mg/L, respectively. Exposure to both coppers considerably decreased pigment levels and photosynthetic efficiency, while inducing the generation of reactive oxygen species (ROS) in cells with increased exposure time. Moreover, the coppers significantly increased the levels of lipid peroxidation and superoxide dismutase (SOD) activity, even at relatively lower concentrations. These suggest that copper contaminants may exert deleterious effects on the photosynthesis and cellular oxidative stress of C. ehrenbergii, representing its powerful potential in aquatic toxicity assessments.

Physiological and biochemical responses of the freshwater green algae Closterium ehrenbergii to the common disinfectant chlorine.

Chlorine (Cl2) is widely used as a disinfectant in water treatment plants and for cleaning swimming pools; it is finally discharged into aquatic environments, possibly causing damage to the non-target organisms in the receiving water bodies. Present study evaluated the effects of the biocide Cl2 to the green alga Closterium ehrenbergii (C. ehrenbergii). Growth rate, chlorophyll a levels, carotenoids, chlorophyll autofluorescence, and antioxidant enzymes were monitored up to 72-h after Cl2 exposure. C. ehrenbergii showed dose-dependent decrease in growth rate and cell division after exposure to Cl2. By using cell counts, the median effective concentration (EC50)-72-h was calculated to be 0.071mgL(-1). Cl2 significantly decreased the pigment levels and chlorophyll autofluorescence intensity, indicating that the photosystem was damaged in C. ehrenbergii. In addition, it increased the production of reactive oxygen species (ROS) in the cells. This stressor significantly increased the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase, and glutathione, and affected the physiology of the cells. These results indicate that Cl2 induces oxidative stress in the cellular metabolic process and leads to physiological and biochemical damages in the green algae. Cl2 discharged in industrial effluents and from water treatment plants may cause harmful effects to the C. ehrenbergii a common freshwater microalgae and other non-target organisms.

Effects of Biocide Chlorine on Biochemical Responses of the Dinoflagellate Prorocentrum minimum.

Effects of the biocide sodium hypochlorite (NaOCl) on the dinoflagellate Prorocentrum minimum were assessed. Growth rate, pigment concentrations, and chlorophyll autofluorescence were monitored up to 72 hours after NaOCl exposure, and these parameters showed dose- and time-dependent decrease. The 72-hour EC50 was 0.983 mg/L. Additionally, enzymatic activities of lipid peroxidation and reduced glutathione were significantly altered with increasing NaOCl and exposure time. Thus, NaOCl at doses of 0.5 mg/L induces physiological and biochemical changes in P. minimum, suggesting that chlorine concentrations observed in power plant discharges and in drinking water systems are potentially detrimental to microalgae.

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR.

The green alga Closterium ehrenbergii occurs in fresh water environments and has been suggested as a model for ecotoxicological assessment. Quantitative real-time PCR (qRT-PCR), with its high sensitivity and specificity, is a preferred method for reliable quantification of gene expression levels. qRT-PCR requires reference genes to normalize the transcription level of the target gene, and selection of appropriate references is crucial. Here, we evaluated nine housekeeping genes, that is, 18S rRNA, ACT, TUA, TUB, eIF, H4, UBQ, rps4, and GAPDH, using 34 RNA samples of C. ehrenbergii cultured in various environments (e.g. exposure to heat shock, UV, metals, and non-metallic chemicals). Each housekeeping gene tested displayed different ranges of C T values for each experimental condition. The gene stability was determined using the descriptive statistic software geNorm, which showed that ACT, H4, and TUA were the most suitable reference genes for all the conditions tested. In addition, at least three genes were required for proper normalization. With these references, we assessed the expression level of the heat shock protein 70 (HSP70) gene in C. ehrenbergii cells exposed to thermal and toxic contaminant stress and found that it was significantly up-regulated by these stressors. This study provides potential reference genes for gene expression studies on C. ehrenbergii with qRT-PCR.

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress.

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5'UTR, and 194-bp 3'UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.

Physiological and biochemical responses of the marine dinoflagellate Prorocentrum minimum exposed to the oxidizing biocide chlorine.

Toxic effects of the commonly used biocide chlorine (Cl2) on the marine dinoflagellate Prorocentrum minimum were assessed using growth-, pigment- and enzyme activity-based endpoints. Cell count, chlorophyll a levels, carotenoids, and chlorophyll autofluorescence were monitored up to 72h after exposure to Cl2, and these parameters showed a dose- and time-dependent decrease. The 72-h median effective concentration (EC50) based on growth rate was 1.177mgL(-1). Cl2 dose above 0.5mgL(-1) were toxic to P. minimum after 6-h exposure to Cl2; the effect increased with increase in exposure time as revealed by a significant reduction in growth rate and decreased chlorophyll fluorescence. Moreover, the activities of antioxidant enzymes, including superoxide dismutase and catalase, were altered proportionally with increasing Cl2 dose. The results of this study show that Cl2 concentrations as observed in power-plant discharges and in drinking-water systems cause physiological and biochemical damage to the microalgae.

Quantification of toxic effects of the herbicide metolachlor on marine microalgae Ditylum brightwellii (Bacillariophyceae), Prorocentrum minimum (Dinophyceae), and Tetraselmis suecica (Chlorophyceae).

Toxic effects of the herbicide metolachlor (MC) were evaluated for three marine microalgae, Tetraselmis suecica (chlorophyte), Ditylum brightwellii (diatom), and Prorocentrum minimum (dinoflagellate). MC showed a significant reduction in cell counts and chlorophyll a levels. Median effective concentration (EC50) was calculated based on chlorophyll a levels after a 72-h MC exposure. EC50 values for T. suecica, D. brightwellii, and P. minimum were 21.3, 0.423, and 0.07 mg/L, respectively. These values showed that the dinoflagellate was most sensitive when exposed to the herbicide, at a concentration comparable to freshwater algae, suggesting its potential as an appropriate model organism for ecotoxicity assessments in marine environments.

Chlorination-induced cellular damage and recovery in marine microalga, Chlorella salina.

Power plants employ chlorination for controlling biofouling in the cooling water system. Phytoplankton drawn into the cooling water system could be impacted by chemical stress induced by the oxidizing biocide. It is likely that microalgae, being sensitive to chlorine, could suffer damage to their cellular structure and function. In this study, we present data on the effect of in-use concentrations of chlorine on the unicellular microalga, Chlorella salina. Chlorophyll autofluorescence was measured in terms of mean fluorescence intensity per cell for rapid assessment of toxicity. Viability of the cells exposed to chlorine was determined by fluorescein diacetate staining. Functionality of the photosynthetic machinery was assessed by gross primary productivity. Results from the study, which combined confocal laser scanning microscopy with image analysis, showed a significant dose-dependant reduction in chlorophyll autofluorescence, esterase activity and gross primary productivity in chlorine-treated cells. Interestingly, the cells injured by chlorination could not recover in terms of autofluorescence, esterase activity or productivity even after 18 h incubation in healthy media. Among the test points evaluated, esterase activity appeared to be sensitive for determining the chlorination-induced impact. Our results demonstrate that low-dose chlorination causes significant decrease in chlorophyll autofluorescence, intracellular esterase activity and primary productivity in Chlorella cells.

Molecular detection, quantification, and diversity evaluation of microalgae.

This study reviews the available molecular methods and new high-throughput technologies for their practical use in the molecular detection, quantification, and diversity assessment of microalgae. Molecular methods applied to other groups of organisms can be adopted for microalgal studies because they generally detect universal biomolecules, such as nucleic acids or proteins. These methods are primarily related to species detection and discrimination among various microalgae. Among current molecular methods, some molecular tools are highly valuable for small-scale detection [e.g., single-cell polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and biosensors], whereas others are more useful for large-scale, high-throughput detection [e.g., terminal restriction length polymorphism, isothermal nucleic acid sequence-based amplification, loop-mediated isothermal amplification, microarray, and next generation sequencing (NGS) techniques]. Each molecular technique has its own strengths in detecting microalgae, but they may sometimes have limitations in terms of detection of other organisms. Among current technologies, qPCR may be considered the best method for molecular quantification of microalgae. Metagenomic microalgal diversity can easily be achieved by 454 pyrosequencing rather than by the clone library method. Current NGS, third and fourth generation technologies pave the way for the high-throughput detection and quantification of microalgal diversity, and have significant potential for future use in field monitoring.