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.

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.

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.

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.

Studies on chloroplast development and replication in Euglena. I. Vitamin B12 and chloroplast replication.

When Euglena gracilis is grown under vitamin B(12) deficiency conditions, the amount of protein and of chlorophyll per cell increase with decrease of B(12) in the medium and consequently in the cell. The increase in cell protein is proportional to and precedes an increase in the number of chloroplasts per cell. This replication of the chloroplasts under deficiency conditions is not accompanied by nuclear or cell division. It is concluded that chloroplast replication in Euglena gracilis is independent of nuclear and cellular replication, at least under B(12) deficiency conditions. We established a graph of the growth of Euglena under different concentrations of vitamin B(12) added to the growth medium, which permitted us to calculate that at least 22,000 molecules of vitamin B(12) per cell are required to give normal growth.

Freeze etching of cells without cryoprotectants.

The technique of spray-freeze etching was applied to unicellular organisms. The superior freezing rates obtainable with this method gave excellent cryofixation on Chlorella, Euglena, and spermatozoa without the use of antifreeze agents, and cell damage due to ice crystal formation was never observed. In many instances the resultant morphology differed significantly from that obtained from glycerol-treated, freeze-etched cells. Furthermore, viability studies of spray-frozen Chlorella compared favorably with cells frozen by other methods.

Euglena gracilis: formation of giant mitochondria.

The addition of antimycin A (0.5 microgram per milliliter) to cultures of a bleached strain of Euglena gracilis in the logarithmic phase of growth on succinate as a carbon source results in (i) an interruption of growth for 24 hours and (ii) an increase in whole-cell respiration and the emergence of a novel succinoxidase activity within 2 to 4 hours. After 3 to 5 hours, the mitochondria enlarge, fuse, and form a sheathlike structure situated close to the periphery of the cell.

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.

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).

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.

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.

Activities of various cobalamins for Euglena gracilis with reference to vitamin B 12 assay with Euglena.

Coenzyme B(12) and methylcobalamin in water are less active in promoting growth of Euglena gracilis Z strain than the same concentrations of cyanocobalamin and hydroxocobalamin which are equally active. When bound to human serum or human liver homogenate, however, the activities of these four cobalamins do not differ significantly with one exception. The results suggest that the Euglena assay using cyanocobalamin standards is not satisfactory for quantitation of coenzyme B(12) and methylcobalamin in water but acceptable when coenzyme B(12) and methylcobalamin are bound to serum or liver. Sulphitocobalamin in water is as active as cyanocobalamin and hydroxocobalamin but nitritocobalamin is less active. Factor B, the monocarboxylic acids of cyanocobalamin and hydroxocobalamin, and the dicarboxylic acid of cyanocobalamin in water were inactive.

Effect of antimicrobial agents on the Euglena method of serum vitamin B12 assay.

Antimicrobial agents in the serum may affect the results of the Euglena method of serum vitamin B(12) assay. Sulphonamides suppress the growth of Euglena in concentrations attainable in the serum during treatment; streptomycin, chlortetracycline, erythromycin, kanamycin, and nitrofurantoin bleach Euglena but only when present in concentrations far exceeding the normal peak therapeutic blood levels. False low results of serum vitamin B(12) assay due to inhibitory and/or bleaching substances in the serum can be readily detected by microscopy of the assay cultures and Euglena cell counts.

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.

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.

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.