Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Evolutionary Origin of Euglena.

Euglenids (Excavata, Discoba, Euglenozoa, Euglenida) is a group of free-living, single-celled flagellates living in the aquatic environments. The uniting and unique morphological feature of euglenids is the presence of a cell covering called the pellicle. The morphology and organization of the pellicle correlate well with the mode of nutrition and cell movement. Euglenids exhibit diverse modes of nutrition, including phagotrophy and photosynthesis. Photosynthetic species (Euglenophyceae) constitute a single subclade within euglenids. Their plastids embedded by three membranes arose as the result of a secondary endosymbiosis between phagotrophic eukaryovorous euglenid and the Pyramimonas-related green alga. Within photosynthetic euglenids three evolutionary lineages can be distinguished. The most basal lineage is formed by one mixotrophic species, Rapaza viridis. Other photosynthetic euglenids are split into two groups: predominantly marine Eutreptiales and freshwater Euglenales. Euglenales are divided into two families: Phacaceae, comprising three monophyletic genera (Discoplastis, Lepocinclis, Phacus) and Euglenaceae with seven monophyletic genera (Euglenaformis, Euglenaria, Colacium, Cryptoglena, Strombomonas, Trachelomonas, Monomorphina) and polyphyletic genus Euglena. For 150 years researchers have been studying Euglena based solely on morphological features what resulted in hundreds of descriptions of new taxa and many artificial intra-generic classification systems. In spite of the progress towards defining Euglena, it still remains polyphyletic and morphologically almost undistinguishable from members of the recently described genus Euglenaria; members of both genera have cells undergoing metaboly (dynamic changes in cell shape), large chloroplasts with pyrenoids and monomorphic paramylon grains. Model organisms Euglena gracilis Klebs, the species of choice for addressing fundamental questions in eukaryotic biochemistry, cell and molecular biology, is a representative of the genus Euglena.

Biochemistry and Physiology of Reactive Oxygen Species in Euglena.

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are by-products of various metabolic processes in aerobic organisms including Euglena. Chloroplasts and mitochondria are the main sites of ROS generation by photosynthesis and respiration, respectively, through the active electron transport chain. An efficient antioxidant network is required to maintain intracellular ROS pools at optimal conditions for redox homeostasis. A comparison with the networks of plants and animals revealed that Euglena has acquired some aspects of ROS metabolic process. Euglena lacks catalase and a typical selenocysteine containing animal-type glutathione peroxidase for hydrogen peroxide scavenging, but contains enzymes involved in ascorbate-glutathione cycle solely in the cytosol. Ascorbate peroxidase in Euglena, which plays a central role in the ascorbate-glutathione cycle, forms a unique intra-molecular dimer structure that is related to the recognition of peroxides. We recently identified peroxiredoxin and NADPH-dependent thioredoxin reductase isoforms in cellular compartments including chloroplasts and mitochondria, indicating the physiological significance of the thioredoxin system in metabolism of ROS. Besides glutathione, Euglena contains the unusual thiol compound trypanothione, an unusual form of glutathione involving two molecules of glutathione joined by a spermidine linker, which has been identified in pathogenic protists such as Trypanosomatida and Schizopyrenida. Furthermore, in contrast to plants, photosynthesis by Euglena is not susceptible to hydrogen peroxide because of resistance of the Calvin cycle enzymes fructose-1,6-bisphosphatse, NADP+-glyceraldehyde-3-phosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase to hydrogen peroxide. Consequently, these characteristics of Euglena appear to exemplify a strategy for survival and adaptation to various environmental conditions during the evolutionary process of euglenoids.

Biochemistry and Physiology of Vitamins in Euglena.

Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

Biochemistry and Physiology of Heavy Metal Resistance and Accumulation in Euglena.

Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.

Euglena Transcript Processing.

RNA transcript processing is an important stage in the gene expression pathway of all organisms and is subject to various mechanisms of control that influence the final levels of gene products. RNA processing involves events such as nuclease-mediated cleavage, removal of intervening sequences referred to as introns and modifications to RNA structure (nucleoside modification and editing). In Euglena, RNA transcript processing was initially examined in chloroplasts because of historical interest in the secondary endosymbiotic origin of this organelle in this organism. More recent efforts to examine mitochondrial genome structure and RNA maturation have been stimulated by the discovery of unusual processing pathways in other Euglenozoans such as kinetoplastids and diplonemids. Eukaryotes containing large genomes are now known to typically contain large collections of introns and regulatory RNAs involved in RNA processing events, and Euglena gracilis in particular has a relatively large genome for a protist. Studies examining the structure of nuclear genes and the mechanisms involved in nuclear RNA processing have revealed that indeed Euglena contains large numbers of introns in the limited set of genes so far examined and also possesses large numbers of specific classes of regulatory and processing RNAs, such as small nucleolar RNAs (snoRNAs). Most interestingly, these studies have also revealed that Euglena possesses novel processing pathways generating highly fragmented cytosolic ribosomal RNAs and subunits and non-conventional intron classes removed by unknown splicing mechanisms. This unexpected diversity in RNA processing pathways emphasizes the importance of identifying the components involved in these processing mechanisms and their evolutionary emergence in Euglena species.

Photo and Nutritional Regulation of Euglena Organelle Development.

Euglena can use light and CO2, photosynthesis, as well as a large variety of organic molecules as the sole source of carbon and energy for growth. Light induces the enzymes, in this case an entire organelle, the chloroplast, that is required to use CO2 as the sole source of carbon and energy for growth. Ethanol, but not malate, inhibits the photoinduction of chloroplast enzymes and induces the synthesis of the glyoxylate cycle enzymes that comprise the unique metabolic pathway leading to two carbon, ethanol and acetate, assimilation. In resting, carbon starved cells, light mobilizes the degradation of the storage carbohydrate paramylum and transiently induces the mitochondrial proteins required for the aerobic metabolism of paramylum to provide the carbon and energy required for chloroplast development. Other mitochondrial proteins are degraded upon light exposure providing the amino acids required for the synthesis of light induced proteins. Changes in protein levels are due to increased and decreased rates of synthesis rather than changes in degradation rates. Changes in protein synthesis rates occur in the absence of a concomitant increase in the levels of mRNAs encoding these proteins indicative of photo and metabolic control at the translational rather than the transcriptional level. The fraction of mRNA encoding a light induced protein such as the light harvesting chlorophyll a/b binding protein of photosystem II, (LHCPII) associated with polysomes in the dark is similar to the fraction associated with polysomes in the light indicative of photoregulation at the level of translational elongation. Ethanol, a carbon source whose assimilation requires carbon source specific enzymes, the glyoxylate cycle enzymes, represses the synthesis of chloroplast enzymes uniquely required to use light and CO2 as the sole source of carbon and energy for growth. The catabolite sensitivity of chloroplast development provides a mechanism to prioritize carbon source utilization. Euglena uses all of its resources to develop the metabolic capacity to utilize carbon sources such as ethanol which are rarely in the environment and delays until the rare carbon source is no longer available forming the chloroplast which is required to utilize the ubiquitous carbon source, light and CO2.

Protein Targeting to the Plastid of Euglena.

The lateral transfer of photosynthesis between kingdoms through endosymbiosis is among the most spectacular examples of evolutionary innovation. Euglena, which acquired a chloroplast indirectly through an endosymbiosis with a green alga, represents such an example. As with other endosymbiont-derived plastids from eukaryotes, there are additional membranes that surround the organelle, of which Euglena has three. Thus, photosynthetic genes that were transferred from the endosymbiont to the host nucleus and whose proteins are required in the new plastid, are now faced with targeting and plastid import challenges. Early immunoelectron microscopy data suggested that the light-harvesting complexes, photosynthetic proteins in the thylakoid membrane, are post-translationally targeted to the plastid via the Golgi apparatus, an unexpected discovery at the time. Proteins targeted to the Euglena plastid have complex, bipartite presequences that direct them into the endomembrane system, through the Golgi apparatus and ultimately on to the plastid, presumably via transport vesicles. From transcriptome sequencing, dozens of plastid-targeted proteins were identified, leading to the identification of two different presequence structures. Both have an amino terminal signal peptide followed by a transit peptide for plastid import, but only one of the two classes of presequences has a third domain-the stop transfer sequence. This discovery implied two different transport mechanisms; one where the protein was fully inserted into the lumen of the ER and another where the protein remains attached to, but effectively outside, the endomembrane system. In this review, we will discuss the biochemical and bioinformatic evidence for plastid targeting, discuss the evolution of the targeting system, and ultimately provide a working model for the targeting and import of proteins into the plastid of Euglena.

Gravitaxis in Euglena.

Motile microorganisms utilize a number of responses to external stimuli including light, temperature, chemicals as well as magnetic and electric fields. Gravity is a major clue to select a niche in their environment. Positive gravitaxis leads an organism down into the water column and negative gravitaxis brings it to the surface. In Euglena the precision of gravitaxis is regulated by an internal rhythm entrained by the daily light/dark cycle. This and the cooperation with phototaxis bring the cells into an optimal position in the water column. In the past a passive orientation based on a buoy mechanism has been proposed for Euglena gracilis, but now it has been proven that this flagellate possesses a physiological gravireceptor and an active orientation. Numerous experiments in space using satellites, rockets and shuttles as well as in parabolic flights have been conducted as well as in functional weightlessness (simulated microgravity) on ground-based facilities such as clinostats to characterize the gravitaxis of Euglena. The threshold for gravity perception was determined and physiological, biochemical and molecular components of the signal transduction chain have been identified. In contrast to higher plants, some algae and ciliates, Euglena does not possess sedimenting statoliths to detect the direction of the gravity vector of the Earth. The gravireceptors were found to be mechano-sensitive Ca2+-conducting ion channels thought to be located at the front end of the cell underneath the trailing flagellum. When activated by gravity-induced pressure due to sedimentation of the whole cell body, they allow a passive influx of calcium along a previously established ion gradient. The entering calcium binds to a specific calmodulin (CaM.2) which in turn activates an adenylyl cyclase producing cAMP from ATP. This cAMP is believed to activate a specific protein kinase A (PK.4), which is postulated to phosphorylate proteins inside the flagellum resulting in a bending and thus a course correction and reorientation with respect to the direction of the gravity vector. The elements of the signal transduction chain have been characterized by inhibitors and by RNAi to prove their involvement in gravitaxis.

Fast phase processing in off-axis holography by CUDA including parallel phase unwrapping.

We present parallel processing implementation for rapid extraction of the quantitative phase maps from off-axis holograms on the Graphics Processing Unit (GPU) of the computer using computer unified device architecture (CUDA) programming. To obtain efficient implementation, we parallelized both the wrapped phase map extraction algorithm and the two-dimensional phase unwrapping algorithm. In contrast to previous implementations, we utilized unweighted least squares phase unwrapping algorithm that better suits parallelism. We compared the proposed algorithm run times on the CPU and the GPU of the computer for various sizes of off-axis holograms. Using the GPU implementation, we extracted the unwrapped phase maps from the recorded off-axis holograms at 35 frames per second (fps) for 4 mega pixel holograms, and at 129 fps for 1 mega pixel holograms, which presents the fastest processing framerates obtained so far, to the best of our knowledge. We then used common-path off-axis interferometric imaging to quantitatively capture the phase maps of a micro-organism with rapid flagellum movements.

Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday.

A biotic game design project for integrated life science and engineering education.

Engaging, hands-on design experiences are key for formal and informal Science, Technology, Engineering, and Mathematics (STEM) education. Robotic and video game design challenges have been particularly effective in stimulating student interest, but equivalent experiences for the life sciences are not as developed. Here we present the concept of a "biotic game design project" to motivate student learning at the interface of life sciences and device engineering (as part of a cornerstone bioengineering devices course). We provide all course material and also present efforts in adapting the project's complexity to serve other time frames, age groups, learning focuses, and budgets. Students self-reported that they found the biotic game project fun and motivating, resulting in increased effort. Hence this type of design project could generate excitement and educational impact similar to robotics and video games.

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.

Electrofluidics fabricated by space-selective metallization in glass microfluidic structures using femtosecond laser direct writing.

Space-selective metallization of the inside of glass microfluidic structures using femtosecond laser direct-write ablation followed by electroless plating is demonstrated. Femtosecond laser direct writing followed by thermal treatment and successive chemical etching allows us to fabricate three-dimensional microfluidic structures inside photosensitive glass. Then, femtosecond laser ablation followed by electroless metal plating enables flexible deposition of patterned metal films on desired locations of not only the top and bottom walls but also the sidewalls of fabricated microfluidic structures. A volume writing scheme for femtosecond laser irradiation inducing homogeneous ablation on the sidewalls of microfluidic structures is proposed for sidewall metallization. The developed technique is used to fabricate electrofluidics in which microelectric components are integrated into glass microchannels. The fabricated electrofluidics are applied to control the temperature of liquid samples in the microchannels for the enhancement of chemical reactions and to manipulate the movement of biological samples in the microscale space.

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.

Circadian clocks and cell division: what's the pacemaker?

Evolution has selected a system of two intertwined cell cycles: the cell division cycle (CDC) and the daily (circadian) biological clock. The circadian clock keeps track of solar time and programs biological processes to occur at environmentally appropriate times. One of these processes is the CDC, which is often gated by the circadian clock. The intermeshing of these two cell cycles is probably responsible for the observation that disruption of the circadian system enhances susceptibility to some kinds of cancer. The core mechanism underlying the circadian clockwork has been thought to be a transcription & translation feedback loop (TTFL), but recent evidence from studies with cyanobacteria, synthetic oscillators and immortalized cell lines suggests that the core circadian pacemaking mechanism that gates cell division in mammalian cells could be a post-translational oscillator (PTO).

Circadian clocks and antiaging: do non-aging microalgae like Euglena reveal anything?

Microalgae that divide symmetrically in all aspects do not age. While the evolutionary reason for this is obvious, little attention has been paid to the mechanistic explanations. A great deal of study involving many research fields would be needed to explain the mechanisms if we suppose that the immortality results from a lifelong sufficiency of defense from stress or from an essential part of counteracting age-accompanied damage accumulation. Additionally, little is known about the relationships between homeostasis and circadian clocks in antiaging, although each of these has been studied separately. Here, we present a conceptual generalization of those relationships, as suggested by evidence from non-aging microalgae, mainly Euglena. The circadian gating of mitosis and circadian temporal coordination may respectively reduce radiation- and disharmony-induced stress in which homeostasis cannot be involved, whereas circadian resistance rhythms may greatly help homeostatic defense from radiation- and metabolism-induced stress. We also briefly sketch mammalian aging research to compare the current status of knowledge with that of algal antiaging.

Relationship of protozoan biomass to phosphate and nitrate removal from activated sludge mixed liquor.

The relationship between protozoan biomass concentration and phosphate and nitrate removal was investigated in mixed liquor using three different carbon sources as supplements. The study was carried out using three respective initial biomass concentrations in a shaking flask environment. Samples were taken every 24 h to determine phosphate, nitrate, dissolved oxygen and chemical oxygen demand. The results revealed a direct relationship between decreases in nutrient concentrations and increases in cell densities of the isolates. Between 24 and 96 h, the increases in the protozoan density corresponded to a phosphate decreases from initial ranges of 55.42-57.36 mg/L, 50.27-51.17 mg/L and 50.01-50.83 mg/L to final ranges of 2.46-11.90 mg/L, 0.61-11.80 mg/L and 1.29-13.89 mg/L, in the presence of Aspidisca, Trachelophyllum and Peranema, respectively. Nitrate concentrations were observed to decrease from initial ranges of 23.84-25.90 mg/L, 23.94-25.84 mg/L and 26.12-26.54 mg/L to final ranges of 0.11-6.32 mg/L, 0.16-5.60 mg/L and 0.24-9.04 mg/L, respectively. The study had revealed that an increase in cell density of the test isolates produces a corresponding increase in phosphate and nitrate removal.

Photoorientation in photosynthetic flagellates.

Motile microorganisms react to a host of external stimuli, including light, gravity, the magnetic field of the Earth as well as thermal and chemical gradients, in their habitat in order to select a niche suitable for survival and reproduction. Several forms of light-induced behavior have been described in microorganisms including phototaxis, photophobic responses, and photokinesis. Other functions of photoreceptors are regulation of development and entrainment of circadian rhythms. Basically five types of photoreceptor molecules have been identified in microorganisms: BLUF proteins, cryptochromes, phototropins, phytochromes, and rhodopsins. The photoreceptors can control light-activated ion channels or activated enzymes. The responses to the different stimuli in their habitat can be connected in a complex network of signal transduction chains.

Macroevolution via secondary endosymbiosis: a Neo-Goldschmidtian view of unicellular hopeful monsters and Darwin's primordial intermediate form.

Seventy-five years ago, the geneticist Richard Goldschmidt hypothesized that single mutations affecting development could result in major phenotypic changes in a single generation to produce unique organisms within animal populations that he called "hopeful monsters". Three decades ago, Sarah P. Gibbs proposed that photosynthetic unicellular micro-organisms like euglenoids and dinoflagellates are the products of a process now called "secondary endosymbiosis" (i.e., the evolution of a chloroplast surrounded by three or four membranes resulting from the incorporation of a eukaryotic alga by a eukaryotic heterotrophic host cell). In this article, we explore the evidence for Goldschmidt's "hopeful monster" concept and expand the scope of this theory to include the macroevolutionary emergence of organisms like Euglena and Chlorarachnion from secondary endosymbiotic events. We argue that a Neo-Goldschmidtian perspective leads to the conclusion that cell chimeras such as euglenids and dinoflagellates, which are important groups of phytoplankton in freshwater and marine ecosystems, should be interpreted as "successful monsters". In addition, we argue that Charles Darwin had euglenoids (infusoria) in mind when he speculated on the "primordial intermediate form", although his Proto-Euglena-hypothesis for the origin of the last common ancestor of all forms of life is no longer acceptable.