The impact of elevated sulfur and nitrogen levels on cadmium tolerance in Euglena species.

Heavy metal (HM) pollution threatens human and ecosystem health. Current methods for remediating water contaminated with HMs are expensive and have limited effect. Therefore, bioremediation is being investigated as an environmentally and economically viable alternative. Freshwater protists Euglena gracilis and Euglena mutabilis were investigated for their tolerance to cadmium (Cd). A greater increase in cell numbers under Cd stress was noted for E. mutabilis but only E. gracilis showed an increase in Cd tolerance following pre-treatment with elevated concentrations of S or N. To gain insight regarding the nature of the increased tolerance RNA-sequencing was carried out on E. gracilis. This revealed transcript level changes among pretreated cells, and additional differences among cells exposed to CdCl2. Gene ontology (GO) enrichment analysis reflected changes in S and N metabolism, transmembrane transport, stress response, and physiological processes related to metal binding. Identifying these changes enhances our understanding of how these organisms adapt to HM polluted environments and allows us to target development of future pre-treatments to enhance the use of E. gracilis in bioremediation relating to heavy metals.

Determination of hazardous concentrations of 2,4-dinitrophenol in freshwater ecosystems based on species sensitivity distributions.

2,4-dinitrophenol (2,4-DNP) is a phenolic compound used as a wood preservative or pesticide. The chemical is hazardous to freshwater organisms. Although 2,4-DNP poses ecological risks, only a few of its aquatic environmental risks have been investigated and very limited guidelines for freshwater aquatic ecosystems have been established by governments. This study addresses the paucity of 2,4-DNP toxicity data for freshwater ecosystems and the current lack of highly reliable trigger values for this highly toxic compound. We conducted acute bioassays using 12 species from nine taxonomic groups and chronic assays using five species from four taxonomic groups to improve the quality of the dataset and enable the estimation of protective concentrations based on species sensitivity distributions. The acute and hazardous concentrations of 2,4-DNP in 5% of freshwater aquatic species (HC5) were determined to be 0.91 (0.32-2.65) mg/L and 0.22 (0.11-0.42) mg/L, respectively. To the best of our knowledge, this is the first report of a suggested chronic HC5 for 2,4-DNP and it provides the much-needed fundamental data for the risk assessment and management of freshwater ecosystems.

Anthracene phytotoxicity in the freshwater flagellate alga Euglena agilis Carter.

The freshwater flagellate alga Euglena agilis Carter was exposed to the polycyclic aromatic hydrocarbon (PAH) anthracene for 96 h under optimal photosynthetically active radiation (PAR), and responses of growth, photosynthetic pigment production, and photosynthetic efficiency were assessed. Anthracene reduced the growth rate (µ) and levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids. The growth rate was more sensitive than photosynthetic parameters, with a median effective concentration (EC50) of 4.28 mg L-1. Between 5 and 15 mg L-1, anthracene inhibited the maximum quantum yield (Fv/Fm) of photosystem II (PSII) and the maximum photosynthetic electron transport rate through PSII (rETRmax) with EC50 values of 14.88 and 11.8 mg L-1, respectively. At all anthracene concentrations, intracellular reactive oxygen species (ROS) were elevated, indicating increased oxidative stress. Anthracene presumably reduced the PSII efficiency of photochemical energy regulation and altered the photochemistry through intracellular ROS formation. Acute exposure to PAHs may induce severe physiological changes in phytoplankton cells, which may influence vital ecological processes within the aquatic environments. Additionally, growth and Chl a content may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC50 values for both overlap with anthracene levels (8.3 mg L-1) permitted by the US Environmental Protection Agency (USEPA).

The dialogue between protozoa and bacteria in a microfluidic device.

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Arsenic hypertolerance in the protist Euglena mutabilis is mediated by specific transporters and functional integrity maintenance mechanisms.

Arsenic is a toxic metalloid known to cause multiple and severe cellular damages, including lipid peroxidation, protein misfolding, mutagenesis and double and single-stranded DNA breaks. Thus, exposure to this compound is lethal for most organisms but some species such as the photosynthetic protist Euglena mutabilis are able to cope with very high concentrations of this metalloid. Our comparative transcriptomic approaches performed on both an arsenic hypertolerant protist, i.e. E. mutabilis, and a more sensitive one, i.e. E. gracilis, revealed multiple mechanisms involved in arsenic tolerance. Indeed, E. mutabilis prevents efficiently the accumulation of arsenic in the cell through the expression of several transporters. More surprisingly, this protist induced the expression of active DNA reparation and protein turnover mechanisms, which allow E. mutabilis to maintain functional integrity of the cell under challenging conditions. Our observations suggest that this protist has acquired specific functions regarding arsenic and has developed an original metabolism to cope with acid mine drainages-related stresses.

A rapid phenol toxicity test based on photosynthesis and movement of the freshwater flagellate, Euglena agilis Carter.

Phenol, a monosubstituted aromatic hydrocarbon with various commercial uses, is a major organic constituent in industrial wastewaters. The ecotoxic action of phenol for aquatic environment is well known. In this study, rapid phenol toxicity tests (1h) were developed based on chlorophyll a (Chl a) fluorescence and the movement parameters of the freshwater flagellate, Euglena agilis Carter. Phenol significantly reduced the maximum quantum yield (Fv/Fm) of photosystem II (PS II) and the maximum photosynthetic electron transport rate (rETRmax) with median effective concentration (EC50) values of 8.94 and 4.67 mM, respectively. Phenol reduced the motility and triggered change in the swimming velocity of the test organism. Among the parameters tested, velocity was the most sensitive biomarker with an EC50 of 3.17 mM. The EC50 values for Fv/Fm, motility, and velocity appear to overlap the permitted levels of phenol. In conclusion, the photosynthesis and movement of E. agilis can be fast and sensitive risk assessment parameters for the evaluation of phenol toxicity in municipal and industrial effluents.

Gas/liquid sensing via chemotaxis of Euglena cells confined in an isolated micro-aquarium.

We demonstrate on-chip gas/liquid sensing by using the chemotaxis of live bacteria (Euglena gracilis) confined in an isolated micro-aquarium, and gas/liquid permeation through porous polydimethylsiloxane (PDMS). The sensing chip consisted of one closed micro-aquarium and two separated bypass microchannels along the perimeter of the micro-aquarium. Test gas/liquid and reference samples were introduced into the two individual microchannels separately, and the gas/liquid permeated through the PDMS walls and dissolved in the micro-aquarium water, resulting in a chemical concentration gradient in the micro-aquarium. By employing the closed micro-aquarium isolated from sample flows, we succeeded in measuring the chemotaxis of Euglena for a gas substance quantitatively, which cannot be achieved with the conventional flow-type or hydro-gel-type microfluidic devices. We found positive (negative) chemotaxis for CO2 concentrations below (above) 15%, with 64 ppm as the minimum concentration affecting the cells. We also observed chemotaxis for ethanol and H2O2. By supplying culture medium via the microchannels, the Euglena culture remained alive for more than 2 months. The sensing chip is thus useful for culturing cells and using them for environmental toxicity/nutrition studies by monitoring their motion.

Surface properties and intracellular speciation revealed an original adaptive mechanism to arsenic in the acid mine drainage bio-indicator Euglena mutabilis.

Euglena mutabilis is a protist ubiquitously found in extreme environments such as acid mine drainages which are often rich in arsenic. The response of E. mutabilis to this metalloid was compared to that of Euglena gracilis, a protist not found in such environments. Membrane fatty acid composition, cell surface properties, arsenic accumulation kinetics, and intracellular arsenic speciation were determined. The results revealed a modification in fatty acid composition leading to an increased membrane fluidity in both Euglena species under sublethal arsenic concentrations exposure. This increased membrane fluidity correlated to an induced gliding motility observed in E. mutabilis in the presence of this metalloid but did not affect the flagellar dependent motility of E. gracilis. Moreover, when compared to E. gracilis, E. mutabilis showed highly hydrophobic cell surface properties and a higher tolerance to water-soluble arsenical compounds but not to hydrophobic ones. Finally, E. mutabilis showed a lower accumulation of total arsenic in the intracellular compartment and an absence of arsenic methylated species in contrast to E. gracilis. Taken together, our results revealed the existence of a specific arsenical response of E. mutabilis that may play a role in its hypertolerance to this toxic metalloid.

Identification of euglenophycin--a toxin found in certain euglenoids.

Currently cyanoprokaryotic algae, diatoms, haptophytes, dinoflagellates, euglenoids, and rhaphidophytes are known to produce algal toxins. A previous study by the authors reported euglenoid algae producing toxin(s) in aquaculture ponds, with confirmation based on positive fish bioassays following exposure to the isolated clonal algal cultures. Toxicity was observed in euglenoid culture isolates obtained from the pond as well as a clonal, culture collection taxon. Here we provide conclusive evidence for euglenoid toxin production, including HPLC/MS, MS/MS, and NMR analyses of a clonal (non-axenic) isolate of Euglena sanguinea grown in batch culture. Following wet chemical serial fractionation, toxic activity was identified in both the methanol and hexane extracts. These extracts were then purified using HPLC. Bioassay-guided HPLC fractionation of these two extracts demonstrated that a single class of toxic compounds, identical in mass and similar in molecular structure, was produced by this organism. The toxic compounds exhibited a maximal UV absorbance at 238nm and gave diagnostic mass peaks at 306 (MH(+)) and 288 (MH(+)-H(2)O). Unambiguous molecular structural determination was carried out by high field NMR analysis operating in 1- and 2-dimensions. Though a predominant isomer represented the bulk of the toxin, several stereo- and structural isomers were evidenced by NMR, and HPLC/MS. This compound is an alkaloid similar in structure to fire ant venom. The compound exhibits ichthyotoxic, herbicidal and anticancer activity at low ppm to ppb dosages.

[Genotoxicity effect of organic pollutants in Meiliang Bay of Taihu Lake on microalga Euglena gracilis].

Organic pollutant ingredients and content of water samples from Taihu Lake were analyzed by GC-MS. Results showed that Taihu Lake was already contaminated by the organic pollutant, and 15 kinds of targeted organic pollutants were detected. At lower concentrations (1 time), organic pollutants could not have notable effect on the growth of Euglena gracilis, but could increase the content of photosynthetic pigment. At higher concentrations (5, 10 times), organic pollutants restrained the growth of E. gracilis remarkably, and decreased the content of photosynthetic pigment. Activities of SOD and POD increased with the content of organic pollutants. It is indicated that organic pollution could induce activities of antioxidation enzymes in E. gracilis. TOM and TM for the genotoxicity assay increased and DNA damage was found. In higher concentration groups, DNA damage was serious and had an obvious dose-effect relationship. It is indicated that Meiliang bay water may have potential mutagenicity. Comet assay combined with SOD analysis was of value to genotoxic monitoring of polluted water and was a suitable biomarker for organic pollutants in water.

Circadian rhythms of resistance to UV-C and UV-B radiation in Euglena as related to "escape from light" and "resistance to light".

Radiation-induced stress, either from visible or UV light, is strongest at midday. We found that, in the absence of stress or time cues, Euglena gracilis Z was the most resistant to UV-C and UV-B at subjective midday, whether judged from immediate or reproductive survival. The circadian UV-resistance rhythms were free-running in stationary cultures under 1-h light/1-h dark cycles or continuous darkness, indicating that cell-cycle dependent DNA susceptibility to UV was not involved. We moreover examined what was the primary cause of the circadian UV resistance, estimated as the immediate cell survival. The half-maximal lethal dose (LD(50)) of UV-C at subjective midday (the most resistant phase) was 156 J/m(2), which is approximately 3-fold that at subjective midnight. The same was true for UV-B, except the LD(50) was approximately 13-fold that of UV-C. Temperature during UV irradiation had little effect, indicating that survival was not mediated via enzymatic reactions. Non-enzymatic antioxidants were added 5 min before UV irradiation. Dimethylsulfoxide (a hydroxyl radical scavenger) increased survival after UV-B, but had little effect after UV-C; conversely, sodium ascorbate increased survival after UV-C, but not after UV-B. These findings suggest that circadian rhythms of resistance to UVs involve a common mechanism for maximizing non-enzymatic antioxidative capacity at subjective midday, but the specific antioxidants differ.

Euglenoid blooms in the floodplain wetlands of Barak Valley, Assam, North eastern India.

Red blooms of Euglena sp. in the floodplain wetland ecosystems of Barak Valley, Assam, India, were found to be induced by high concentrations of NH3-N, NO3, Fe, Mg and to some extent, PO4, Cu and Zn in their water. The trace elements were rapidly accumulated by the bloom organisms to high levels, whereby their concentrations in the water declined, leading to a collapse of the bloom, which tended to reappear as decomposition again led to the release of the nutrients. The bloom also harboured fairly high density of certain other algae and zooplankton, thereby acting as a sub-system within the wetland ecosystem. The bloom is non-toxic and is exploited as a fish food by the fish-farmers who artificially induce a bloom for augmenting the growth of surface-feeding species of fishes.

Role of cyclic GMP in the mediation of circadian rhythmicity of the adenylate cyclase-cyclic AMP-phosphodiesterase system in Euglena.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are two second messengers that have been proposed to act as a dualistic system in biological regulation. To determine if cGMP plays a role in the mediation of circadian rhythmicity of the adenylate cyclase (AC)-cAMP-phosphodiesterase (PDE) system in the achlorophyllous ZC mutant of the unicellular flagellate Euglena, the levels of cAMP and cGMP were monitored in synchronized cell populations, and the effects of the cGMP analog 8-bromo-cGMP (8-Br-cGMP) and the cGMP inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583) on the activity of AC and PDE, as well as on the level of cAMP, were measured in vivo. A bimodal, 24-hr rhythm of cGMP content was found in both dividing and nondividing cultures in either a 12-hr:12-hr light-dark cycle or constant darkness. The peaks and troughs of the cGMP rhythm occurred 2 hr in advance of those of the cAMP rhythm that has been reported previously. The addition of 8-Br-cGMP at different circadian times increased the cAMP level in vivo by two to five times, whereas LY 83583 reduced the amplitude of the cAMP rhythm so that it disappeared. The effects of 8-Br-cGMP on the activity of AC and PDE were circadian phase-dependent and consistent with the changes in cAMP content. These findings suggest that cGMP may serve as an upstream effector that mediates the cAMP oscillation by regulation of the AC-cAMP-PDE system.

Effects of a first exposure to ethanol on the compositions of neutral and polar lipids in Euglena gracilis Z, taken as a hepatic cell model: equilibration by citrulline-malate.

In comparison to the lipid composition of Euglena cells fed with lactate, a first exposure of the cells to ethanol favors the production of neutral lipids containing mainly unsaturated fatty acids. The ethanol diminishes drastically the proportion of PC and weakly that of PE. In contrast, it increases slightly the proportion of DPG. The ethanol induces important changes in the fatty acid distributions of each lipid class, suggesting modifications of the elongation-desaturation system. On the one hand the proportion of unsaturated fatty acids is increased and, on the other hand, the last double bond is predominantly situated in the delta 6 position in place of delta 3. The addition of the complex citrulline-malate corrects most of these changes.

A genotoxicological study of the new local anaesthetic carbamate derivatives carbisocaine, heptacaine and pentacaine.

The potential mutagenic activity of three carbamate derivatives with local anaesthetic activity was investigated. Genotoxic activity was observed after application of Carbisocaine on Euglena gracilis, whereas no activity was detected by Carbisocaine, Heptacaine and Pentacaine on Salmonella typhimurium, Escherichia coli and Drosophila melanogaster.

Injured mitochondria in cells of Euglena gracilis after DNA gyrase inhibitors treatment.

Five quinolone (ofloxacin, cinoxacin, enoxacin, ciprofloxacin, oxolinic acid) and one non-quinolone (coumermycin A1) inhibitors of prokaryotic DNA gyrase used in clinical practice for treatment of bacterial infections were experimentally examinated. As model organism the flagellate Euglena gracilis was used. Ultrastructural changes in chloroplasts and mitochondria caused by inhibitors were quantitavely evaluated. Simultaneously in all cases injury and hereditary loss of chloroplasts (bleaching) were observed in the cells. In some samples about 45% of cup-shaped mitochondria cumulated in the cytoplasm. In damaged mitochondria some degenerative signs were seen, but after the last subcultivation on drug-free media the number of injured mitochondria in the bleached cells yielded to the normal value.

Algal photosensory apparatus probably represent multiple parallel evolutions.

We postulate that algal photoresponse mechanisms are of relatively recent origin and represent numerous parallel evolutions. Functional differences among them are evident, and it is unlikely that any one can be taken as a "model system" representing all. It is probable that the light antenna is the only truly novel part of the apparatus in most cases, with the signal-processing and motile elements being borrowed from some other, more ancient, sensory system. It is thus anticipated that the light-antennae will show the greatest phylogenetic variation, whereas the signal-processors and motile elements will resemble those of other sensory systems in the same groups of organisms.

Surface properties of the euglenoid flagellum.

New structural details of the Euglena flagellum have led to a modified interpretation of the arrangement of the mastigoneme sheath and its internal attachment. A paraaxial ribbon is described which is located between the flagellar membrane and the axonemal microtubules. This fine ribbon apparently binds mastigoneme units and in turn is bonded to three peripheral microtubule doublets in a position approximately opposite that of the paraflagellar rod. The latter structure seems to anchor one half of the flagellar sheath while the paraaxial ribbon anchors the other one half of the flagellar sheath. Immunological labelling of Euglena mastigonemes has demonstrated that mastigonemes are present in the reservoir as well as on the flagellar surface if monovalent Fab' is used on deflagellated cells. Pulse labelling with anti-mastigoneme Fab' in regenerating cells showed the initial reservoir label was lost and indicated that the labelled mastigonemes were transferred to the flagellum. The reservoir is thus demonstrated to contain a surface pool for flagellar mastigonemes. Flagellar regeneration is partially inhibited irreversibly by the glycoprotein synthesis inhibitor tunicamycin. Experiments with cycloheximide and tunicamycin suggest each antibiotic affects different moieties and that some glycoprotein(s) is limiting to flagellar growth in Euglena. It is postulated that mastigonemes are possible candidates for that rate-limiting component.