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In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research. Graphical abstract

The unique environment of space is characterized by several stress factors, including intense radiation, microgravity, high vacuum and extreme temperatures, among others. These stress conditions individually or in-combination influence genetics and gene regulation and bring potential evolutionary changes in organisms that would not occur under the Earth's gravity regime (1 × g). Thus, space can be explored to support the emergence of new varieties of microbes and plants, that when selected for, can exhibit increased growth and yield, improved resistance to pathogens, enhanced tolerance to drought, low nutrient and disease, produce new metabolites and others. These properties may be more difficult to achieve using other approaches under 1 × g. This review provides an overview of the space microgravity and ionizing radiation conditions that significantly influence organisms. Changes in the genomics, physiology, phenotype, growth and metabolites of organisms in real and simulated microgravity and radiation conditions are illustrated. Results of space biological experiments show that the space environment has significant scientific, technological and commercial potential. Combined these potentials can help address the future of life on Earth, part of goal e of astrobiology.

INTRODUCTION: : A long-term stay of humans in space causes a large number of well-known health problems and changes in protists and plants. Deep space exploration will increase the time humans or rodents will spend in microgravity (µg). Moreover, they are exposed to cosmic radiation, hypodynamia, and isolation. OMICS investigations will increase our knowledge of the underlying mechanisms of µg-induced alterations in vivo and in vitro.AREAS COVERED: : We summarize the findings over the recent 3 years on µg-induced changes in the proteome of protists, plants, rodent and human cells. Considering the thematic orientation of microgravity-related publications in that time frame, we focus on medicine-associated findings such as the µg-induced antibiotic resistance of bacteria, the myocardial consequences of µg-induced calpain activation and the role of MMP13 in osteoarthritis. All these point to the fact that µg is an extreme stressor that could not be evolutionarily addressed on Earth.EXPERT COMMENTARY: : In conclusion, when interpreting µg-experiments, the direct, mostly unspecific stress response, must be distinguished from specific µg-effects. For this reason, recent studies often do not consider single protein findings but place them in the context of protein-protein interactions. This enables an estimation of functional relationships, especially if these are supported by epigenetic and transcriptional data (multi-omics).

The common carp (Cyprinus carpio) is an important livestock in regions of Central Europe, where it is bred in shallow, turbid ponds (mean depth 1 m). A frequent problem observed in this form of aquaculture is hypoxia, which confines the fish close to the surface, preventing them from optimally feeding at the bottom. According to information propagated in textbooks and good practice guidebooks and internet documents, the best solution to that is aeration in the early morning hours, when oxygen concentration is supposed to be minimal. While we wanted to test the feasibility of photovoltaic power for aeration of ponds, we detected that on days with bright sunshine the lowest oxygen concentration occurs in the afternoon at the bottom layer of a shallow pond, and that oxygen distribution is highly influenced by thermal stratification. This formation of layers of different temperatures within the water body inhibits the diffusion of oxygen. A breaking of the stratification effectively increases the oxygen concentration, elevating the overall amount of oxygen in the pond. Consequently, the drop in oxygen concentration at night is less dramatic, and critically low levels of oxygen in deeper zones of the pond are avoided. A comparison between the oxygen distribution and daytime or nighttime aeration showed that on days with bright sunshine it is more effective to force mixing of the entire water column during the day, making the use of a photovoltaic power system feasible. This offers a simple and elegant solution for ponds which are not connected to the power grid.

Note: In lieu of an abstract, this is an excerpt from the first page. Nature provides a unique diversity of primary and secondary metabolites [...]

Pollutants, including synthetic organic materials and heavy metals, are known to adversely affect physiological systems in all animal species studied so far, especially in aquatic ecosystems. Although many individual chemicals can disrupt normal functions, the combined actions of various pollutants are particularly worrying as they can have effects even when each individual chemical is present at concentrations too low to be individually effective. A special approach is currently being given to anthropogenic products called emerging pollutants, including drugs, hormones and by-products of human and animal metabolism which, despite being in very low concentrations, can cause biological effects which differ greatly between species, reflecting differences in exposure pattern, uptake pathways, post-uptake metabolism, accumulation rates and target organ sensitivity. Thus, understanding the effects of pollutants on wildlife and aquatic ecosystems will require a detailed study of many different species representing a wide range of taxa. However, these studies can be substantiated by knowledge gained under more controlled conditions, which may indicate likely mechanisms of action and appropriate endpoint measures. Responses may be exacerbated by interactions between the effects of pollutants, and environmental stressors, such as malnutrition or osmotic stress, and changes in these variables associated with climate change may further exacerbate physiological responses to pollutant burden.

The highly diverse group of microalgae offers a huge variety of bioactive molecules. Manipulation of culture conditions for microalgae allows for maximizing the yield of the desired component. Long-chain polyunsaturated fatty acids are important for the development of the human nervous system. Sterols can diminish cardiovascular disease risk factors. Polysaccharides can modulate the immune system, alleviate allergies, and serve as lubricants for damaged joints. The amino acid profile of microalgae protein is of high quality, and peptides derived from it allow pharmaceutical applications. Pigments such as carotenoids are powerful antioxidants and important for eyesight. Theoretically, yields can further be increased by genetic manipulation.

Brazil is the 5th largest country with the 6th largest population in the world; 25% of its population live along the coast of the Atlantic Ocean. Urban and industrial development was historically not met by the implementation of appropriate sanitation infrastructure and is therefore insufficient today. Additionally, only minimal treatment is required to dispose of sewage via submarine outfalls into the sea. Marine pollution is diverse: domestic sewage carries feces, which can contain potentially harmful microorganisms. Feces are fertilizers, causing eutrophication, and they contain pharmaceuticals and their metabolites. Industrial sewage contains trace metals in large quantities and organic pollutants. Huge amounts of mine tailings are accidentally spilled into rivers far from the coast but reach the sea eventually. Careless handling of products in harbors and during transport leads to unintended release of organic and petrochemical pollutants, fertilizers and microplastic pellets. Heavy rainfall flushes litter from landfills or from unattended trash collection sites into rivers and the sea. This chapter gives an overview over types of pollution occurring in Brazil and a more detailed picture of the pollution situation in two of the areas under most anthropogenic pressure, the Santos Bay Region in Sao Paulo State and Guanabara Bay in Rio de Janeiro State.

All life forms have evolved under the constant force of gravity on Earth and developed ways to counterbalance acceleration load. In space, shear forces, buoyance-driven convection, and hydrostatic pressure are nullified or strongly reduced. When subjected to microgravity in space, the equilibrium between cell architecture and the external force is disturbed, resulting in changes at the cellular and sub-cellular levels (e.g., cytoskeleton, signal transduction, membrane permeability, etc.). Cosmic radiation also poses great health risks to astronauts because it has high linear energy transfer values that evoke complex DNA and other cellular damage. Space environmental conditions have been shown to influence apoptosis in various cell types. Apoptosis has important functions in morphogenesis, organ development, and wound healing. This review provides an overview of microgravity research platforms and apoptosis. The sections summarize the current knowledge of the impact of microgravity and cosmic radiation on cells with respect to apoptosis. Apoptosis-related microgravity experiments conducted with different mammalian model systems are presented. Recent findings in cells of the immune system, cardiovascular system, brain, eyes, cartilage, bone, gastrointestinal tract, liver, and pancreas, as well as cancer cells investigated under real and simulated microgravity conditions, are discussed. This comprehensive review indicates the potential of the space environment in biomedical research.

In a recent publication, Edelmann (Protoplasma 2018; 255,1877-1881) refuted the well-established starch-amyloplast hypothesis of gravitropism in plants. Gravitropic curvatures of shoots and roots were still present after amyloplast-containing tissues (in sheath of vascular bundles and root caps) were dissected. Here, we discuss Edelmann's data in the light of Popper's falsification principle.

Long-term spaceflight needs reliable Biological life support systems (BLSS) to supply astronauts with enough food, fresh air and recycle wasters, but the knowledge about the operation pattern and controlling strategy is rear. For this purpose, a miniaturized enclosed aquatic ecosystem was developed and flown on the Chinese spaceship Shenzhou-8. The system with a total volume of about 60 mL was separated into two chambers by means of a gas transparent membrane. The lower chamber was inoculated with Euglena gracilis cells, and the upper chamber was cultured with Chlorella cells and three snails. After 17.5 days flight, the samples were analyzed. It was found that all snails in the ground module (GM) were alive, while in the flight module (FM) only one snail survived. The total cell numbers, assimilation of nutrients like nitrogen and phosphorus, soluble proteins and carbohydrate contents showed a decrease in FM than in GM. The correlation analysis showed upper chambers of both FM and GM had the same positive and negative correlation factors, while differential correlation was found in lower chambers. These results suggested primary productivity in the enclosed system decreased in microgravity, accompanied with nutrients assimilation. The FM chamber endured lacking of domination species to sustain the system development and GM chamber endured richness in population abundance. These results implied photosynthesis intensity should be reduced to keep the system healthy. More Chlorella but less Euglena might be a useful strategy to sustain system stability. It is the first systematic analysis of enclosed systems in microgravity.

The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

Colistin (polymyxin E) is a membrane-destabilizing antibiotic used against Gram-negative bacteria. We have recently reported that the outer membrane prevents the uptake of antibacterial chlorophyllin into Gram-negative cells. In this study, we used sub-toxic concentrations of colistin to weaken this barrier for a combination treatment of Escherichia coli and Salmonella enterica serovar Typhimurium with chlorophyllin. In the presence of 0.25 &micro, g/mL colistin, chlorophyllin was able to inactivate both bacteria strains at concentrations of 5&ndash, 10 mg/L for E. coli and 0.5&ndash, 1 mg/L for S. Typhimurium, which showed a higher overall susceptibility to chlorophyllin treatment. In accordance with a previous study, chlorophyllin has proven antibacterial activity both as a photosensitizer, illuminated with 12 mW/cm2, and in darkness. Our data clearly confirmed the relevance of the outer membrane in protection against xenobiotics. Combination treatment with colistin broadens chlorophyllin&rsquo, s application spectrum against Gram-negatives and gives rise to the assumption that chlorophyllin together with cell membrane-destabilizing substances may become a promising approach in bacteria control. Furthermore, we demonstrated that colistin acts as a door opener even for the photodynamic inactivation of colistin-resistant (mcr-1-positive) E. coli cells by chlorophyllin, which could help us to overcome this antimicrobial resistance.

Introduction: Microgravity (µg) is an extreme stressor for plants, animals, and humans and influences biological systems. Humans in space experience various health problems during and after a long-term stay in orbit. Various studies have demonstrated structural alterations and molecular biological changes within the cellular milieu of plants, bacteria, microorganisms, animals, and cells. These data were obtained by proteomics investigations applied in gravitational biology to elucidate changes in the proteome occurring when cells or organisms were exposed to real µg (r-µg) and simulated µg (s-µg). Areas covered: In this review, we summarize the current knowledge about the impact of µg on the proteome in plants, animals, and human cells. The literature suggests that µg impacts the proteome and thus various biological processes such as angiogenesis, apoptosis, cell adhesion, cytoskeleton, extracellular matrix proteins, migration, proliferation, stress response, and signal transduction. The changes in cellular function depend on the respective cell type. Expert commentary: This data is important for the topics of gravitational biology, tissue engineering, cancer research, and translational regenerative medicine. Moreover, it may provide new ideas for countermeasures to protect the health of future space travelers.

International audience; Living organisms adapt to changing environments using their amazing flexibility to remodel themselves by a process called evolution. Environmental stress causes selective pressure and is associated with genetic and phenotypic shifts for better modifications, maintenance, and functioning of organismal systems. The natural evolution process can be used in complement to rational strain engineering for the development of desired traits or phenotypes as well as for the production of novel biomaterials through the imposition of one or more selective pressures. Space provides a unique environment of stressors (e.g., weightlessness and high radiation) that organisms have never experienced on Earth. Cells in the outer space reorganize and develop or activate a range of molecular responses that lead to changes in cellular properties. Exposure of cells to the outer space will lead to the development of novel variants more efficiently than on Earth. For instance, natural crop varieties can be generated with higher nutrition value, yield, and improved features, such as resistance against high and low temperatures, salt stress, and microbial and pest attacks. The review summarizes the literature on the parameters of outer space that affect the growth and behavior of cells and organisms as well as complex colloidal systems. We illustrate an understanding of gravity-related basic biological mechanisms and enlighten the possibility to explore the outer space environment for application-oriented aspects. This will stimulate biological research in the pursuit of innovative approaches for the future of agriculture and health on Earth.

We compared the responses of two estuarine phytoplankton communities, one from a temperate (Chubut River estuary (CH), Argentina) and one from a sub-tropical site (Babitonga Bay (BB), Brazil), in a scenario of nutrient enrichment under solar ultraviolet radiation (UVR) exposure. Seawater samples were exposed in microcosms to two nutrients, ambient vs. enriched, and two radiation conditions, with and without UVR, and exposed to solar radiation for 4 days. We evaluated the short- (PSII photochemistry, during 90 min light and 90 min dark cycles, before and after the 4 days of acclimation) and mid-term effects (growth and taxonomic changes) of the phytoplankton communities. Before acclimation, short-term UVR effects were dominant in CH, whereas in BB, nutrient effects prevailed. Such differences were related to the previous light history of the cells and to the ambient nutrient status. After acclimation, an overall improvement of the photosynthetic performance was observed at both sites, either by reducing the relative inhibition or by increasing the recovery of the effective photochemical quantum yield. Interactive effects of UVR and nutrients on growth at CH were antagonistic, while at BB, no differences were observed between the interactive and the sum of effects. Part of the differences in the mid-term observed responses can be attributed to taxonomic changes, with the CH community dominated by diatoms throughout the experiment, but with a shift from a diatom to a flagellate-dominated community in BB. Temperature differences between both sites might have favored higher growth rates and flagellates dominance in BB under the nutrient enriched conditions. Fil: Villafañe, Virginia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Jabalera Cabrerizo, Marco. Universidad de Granada; España Fil: Erzinger, Gilmar S.. Universidade da Região de Joinville; Brasil Fil: Bermejo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Strauch, Sebastian Michael. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Valiñas, Macarena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Helbling, Eduardo Walter. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina

The effect of copper and silver ions on photosynthetic parameters of the flagellate Euglena gracilis was determined. The photosystem II quantum yield (Y(PSII)) and steady state fluorescence (Ft) were measured with a PAM-fluorometer, while delayed fluorescence (DF) was determined by means of a sensitive single-photon camera attached to an image analysis device, which counted the relative number of photons released from the algae in darkness after previous light-stimulation. We found that after 3 h of incubation photosynthesis was affected only in the presence of relatively high concentrations of the used noble metals. The obtained EC50 values for Cu2+ were 505.8 mg L−1 (Y (PSII)), 286.5 mg L−1 (DF), and 491.0 mg L−1 (Ft). The EC50 values for silver ions were 51.9 mg L−1 (Y (PSII)), 51.8 mg L−1 (DF) (values for Ft not determinable). Photosynthesis is far less sensitive against toxic noble metals compared to motility parameters as reported in earlier studies, in part because the organelles of photosynthesis, the chloroplasts, are well protected from external substances within the cell, while flagella, reaching into the surrounding of the cell, are more easily influenced. As chloroplasts are easily accessible by means of non-invasive methods e.g. PAM- fluorometry, they are probably interesting model systems in order to detect effects of substances deep inside the cell. Influence on the chloroplasts is a measure that a germicide entered the cells, diffused through the cytoplasm and the membranous systems and reached compartmentalized enzyme complexes.

Dormant states of organisms are easier to store than the living state because they tolerate larger variations in temperature, light, storage space etc., making them attractive for laboratory culture stocks and also for experiments under special circumstances, especially space flight experiments. Like several other organisms,Euglena gracilisis capable of forming desiccation tolerant resting states in order to survive periods of unfavourable environmental conditions. In earlier experiments it was found that dormantEuglenacells must not become completely desiccated. Some residual moisture is required to ensure recovery of the resting states. To analyse the water demand in recovery ofEuglenaresting states, cells were transferred to a defined amount of cotton wool (0.5 g). Subsequently different volumes of medium (1, 2, 3, 4, 5, 8, 10 and 20 ml) were added in order to supply humidity; a control was set up without additional liquid. Samples were sealed in transparent 50 ml falcon tubes and stored for 9 months under three different conditions:•Constant low light conditions in a culture chamber at 20°C,•In a black box, illuminated with short light emitting diode-light pulses provided by joule thieves and•In darkness in a black box.After 9 months, cells were transferred to fresh medium and cell number, photosynthetic efficiency and movement behavior was monitored over 3 weeks. It was found that cells recovered under all conditions except in the control, where no medium was supplied. Transcription levels of 21 genes were analysed with a Multiplex-polymerase chain reaction. One hour after rehydration five of these genes were found to be up-regulated: ubiquitin, heat shock proteins HSP70, HSP90, the calcium-sensor protein frequenin and a distinct protein kinase, which is involved in gravitaxis. The results indicate a transient general stress response of the cells.

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive Bacillus subtilis 168, the Gram-negative Escherichia coli DH5&alpha, and E. coli strain NR698 which has a deficient outer membrane, simulating a Gram-negative &ldquo, without&rdquo, its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm2, B. subtilis showed high sensitivity already at low chlorophyllin concentrations (&le, 105 cfu/mL: &le, 0.1 mg/L, 106&ndash, 108 cfu/mL: 0.5 mg/L), whereas E. coli DH5&alpha, was less sensitive (&le, 105 cfu/mL: 2.5 mg/L, 106 cfu/mL: 5 mg/L, 107&ndash, 108 cfu/mL: ineffective at &le, 25 mg/L chlorophyllin). E. coli NR698 was almost as sensitive as B. subtilis against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, E. coli NR698 and B. subtilis can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.

The unicellular freshwater flagellate Euglena gracilis shows negative gravitactic behavior. Previous experiments have revealed that the orientation is most likely an active physiological process in which the beating pattern of the flagellum is controlled by gravity and mediated by a change in the calcium concentration inside the cell. In a signal transduction chain, the calcium signal activates a calmodulin, which in turn raises the concentration of cAMP. This alters the beating pattern of the flagellum; reorientation is therefore not a passive process driven by buoyancy. In a recent parabolic flight experiment (ESA 45th parabolic flight campaign), we observed the beating of the flagellum with a high-resolution light microscope. Transition from hyper g to microg as well as from microg to hyper g caused a change of the beating pattern of the flagellum, which confirmed the physiological nature of the process. In microg cells stopped moving the flagellum or tried to reorient, while in hyper g, the cells realigned consecutively. The reaction times for the flagellar responses in previous experiments are confirmed.

The motile behavior of the unicellular photosynthetic flagellate Euglena gracilis was studied during a two-week mission on the Russian satellite Foton M2. The precision of gravitactic orientation was high before launch and, as expected, the cells were unoriented during microgravity. While after previous short-term TEXUS flights the precision of orientation was as high as before launch, it took several hours for the organisms to regain their gravitaxis. Also the percentage of motile cells and the swimming velocity of the remaining motile cells were considerably lower than in the ground control. In preparatory experiments the flagellate Euglena was shown to produce considerable amounts of photosynthetically generated oxygen. In a coupling experiment in a prototype for a planned space mission on Foton M3, the photosynthetic producers were shown to supply sufficient amounts of oxygen to a fish compartment with 35 larval cichlids, Oreochromis mossambicus.

The unicellular photosynthetic freshwater flagellate Euglena gracilis is a promising candidate as an oxygen producer in biological life-support systems. In this study, the capacity of Euglena gracilis to cope with different light regimes was determined. Cultures of Euglena gracilis in closed bioreactors were exposed to different dark-light cycles (40 W/m2 light intensity on the surface of the 20 L reactor; cool white fluorescent lamps in combination with a 100 W filament bulb): 1 h–1 h, 2 h–2 h, 4 h–4 h, 6 h–6 h, and 8 h–16 h, respectively. Motility and oxygen development in the reactors were measured constantly. It was found that, during exposure to light-dark cycles of 1 h–1 h, 2 h–2 h, 4 h–4 h, and 6 h–6 h, precision of gravitaxis as well as the number of motile cells increased during the dark phase, while velocity increased in the light phase. Oxygen concentration did not yet reach a plateau phase. During dark-light cycles of 8 h–16 h, fast changes of movement behavior in the cells were detected. The cells showed an initial decrease of graviorientation after onset of light and an increase after the start of the dark period. In the course of the light phase, graviorientation increased, while motility and velocity decreased after some hours of illumination. In all light profiles, Euglena gracilis was able to produce sufficient oxygen in the light phase to maintain the oxygen concentration above zero in the subsequent dark phase. Key Words: Euglena gracilis—Bioreactor—Light-dark cycle—Motility—Gravitaxis. Astrobiology 14, 848–858.

Proteomics is performed in microgravity research in order to determine protein alterations occurring qualitatively and quantitatively, when single cells or whole organisms are exposed to real or simulated microgravity. To this purpose, antibody-dependent (Western blotting, flow cytometry, Luminex(®) technology) and antibody-independent (mass spectrometry, gene array) techniques are applied. The anticipated findings will help to understand microgravity-specific behavior, which has been observed in bacteria, as well as in plant, animal and human cells. To date, the analyses revealed that cell cultures are more sensitive to microgravity than cells embedded in organisms and that proteins changing under microgravity are highly interactive. Furthermore, one has to distinguish between primary gravity-induced and subsequent interaction-dependent changes of proteins, as well as between direct microgravity-related effects and indirect stress responses. Progress in this field will impact on tissue engineering and medicine and will uncover possibilities of counteracting alterations of protein expression at lowered gravity.

In recent times Euglena gracilis Z was employed as primary producer in closed environmental life-support system (CELSS), e.g. in space research. The photosynthetic unicellular flagellate is not capable of utilizing nitrate, nitrite, and urea as nitrogen source. Therefore, ammonium is supplied as an N-source in the lab (provided as diammonium-dihydrogenphosphate, (NH4)2HPO4) to E. gracilis cultures. While nitrate exerts low toxicity to organisms, ammonium is harmful for many aquatic organisms especially, at high pH-values, which causes the ionic NH4+ (low toxicity) to be partially transformed into the highly toxic ammonia, NH3. In earlier reports, Euglena gracilis was described to grow with various amino acids as sole N-source. Our aim was to investigate alternatives for (NH4)2HPO4 as N-source with lower toxicity for organisms co-cultivated with Euglena in a CELSS. The growth kinetics of Euglena gracilis cultures was determined in the presence of different amino acids (glycine, glutamine, glutamic acid, leucine, and threonine). In addition, uptake of those amino acids by the cells was measured. Cell growth in the presence of glycine and glutamine was quite comparable to the growth in (NH4)2HPO4 containing cultures while a delay in growth was observed in the presence of leucine and threonine. Unlike, aforementioned amino acids glutamate consumption was very poor. Cell density and glutamate concentration were almost unaltered throughout the experiment and the culture reached the stationary phase within 8 days. The data are compared with earlier studies in which utilization of amino acids in Euglena gracilis was investigated. All tested amino acids (glutamate with limitations) were found to have the potential of being an alternative N-source for Euglena gracilis. Hence, these amino acids can be used as a non-toxic surrogate for (NH4)2HPO4.

Water soluble chlorophyll (chlorophyllin) exerts pronounced photodynamic activity. Chlorophyllin is a potential remedy against mosquito larvae and aquatic stages in the life cycle of parasites as well as against ectoparasites in fish. In the recent years it was found that mosquito larvae and other pest organisms can be killed by means of photodynamic substances such as different porphyrin derivates (e.g. hematoporphyrin, meso-tri(N-methylpyridyl), meso-mono(N-tetra-decylpyridyl) porphyrine, hematoporphyrin IX, or hermatoporphyrin formula (HPF). It was found that incubation of mosquito larvae in chlorophyllin solution and subsequent irradiation results in photodynamic destruction of the larvae. Incorporation of about 8 ng chlorophyllin per larvae was sufficient to induce its death. In fish mass cultivation ichthyophthiriosis is a severe parasitic protozoan disease caused by the ciliate Ichthyophthirius multifiliis. It was found that incubation of infected fishes in chlorophyllin and subsequent illumination reduced the number of trophonts significantly (more than 50 %). The fishes were not impaired. Chlorophyllin and other photodynamic substances may become a possible countermeasure against I. multifiliis and other ectoparasites in aquaculture. The effectiveness of chlorophyllin depends on light attenuation in the water body.

We carried out short- and mid-term incubations to assess the combined impact of solar ultraviolet radiation (UVR, 280-400nm) and nutrient addition on tropical phytoplankton communities from BabitongaBay, Southern Brazil. Samples were collected at a station ~ 10km inward from the mouth i.e., Inlet, and incubated in microcosms under solar radiation at the surface of the water, with or without UVR, and under two nutrient conditions (ambient and with addition of macronutrients). Nutrient addition had the largest impact on specific growth rates, almost doubling the growth of algae as compared to ambient conditions. UVR-induced inhibition of the effective photochemical quantum yield (Y) at the initial conditions was higher at ambient nutrient conditions than in samples with nutrient addition, demonstrating the antagonistic effect among the two factors. Inhibition (k) and recovery (r) rates of Y at the beginning of the experiment suggest a high sensitivity of this community, but they were reduced after 4 days of exposure in the microcosms, hinting for the presence of acclimation mechanisms. Also, differences in taxonomic composition, with increasing dominance of diatoms in nutrient-enriched cultures, as compared to the initial time and in ambient nutrient conditions (with higher proportion offlagellates) may account for the observed variability in responses along the time as well as within treatments. Fil: Villafañe, Virginia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Erzinger, Gilmar S.. Universidade da Região de Joinville; Brasil Fil: Strauch, Sebastian Michael. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Helbling, Eduardo Walter. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

In order to prepare and support space experiments, 2D and 3D clinostats are widely applied to study the influence of simulated weightlessness on biological systems. In order to evaluate the results a comparison between the data obtained in simulation experiments and in real microgravity is necessary. We are currently analyzing the gravity-dependent behavior of the protists Paramecium biaurelia (ciliate) and Euglena gracilis (photosynthetic flagellate) on these different experimental platforms. So far, first results are presented concerning the behaviour of Euglena on a 2D fast rotating clinostat and a 3D clinostat as well as under real microgravity conditions (TEXUS sounding rocket flight), of Paramecium on a 2D clinostat and in microgravity. Our data show similar results during 2D and 3D clinorotation compared to real microgravity with respect to loss of orientation (gravitaxis) of Paramecium and Euglena and a decrease of linearity of the cell tracks of Euglena. However, the increase of the mean swimming velocities, especially during 3D clinorotation (Euglena) and 2D clinorotation of Paramecium might indicate a persisting mechanostimulation of the cells. Further studies including long-term 2D and 3D clinostat exposition will enable us to demonstrate the qualification of the applied simulation methods.

The German Aerospace Center (DLR) enabled German participation in the joint space campaign on the unmanned Shenzhou 8 spacecraft in November 2011. In this report, the effect of microgravity on Euglena gracilis cells is described. Custom-made dual compartment cell fixation units (containing cells in one chamber and fixative - RNA lysis buffer - in another one) were enclosed in a small container and placed in the Simbox incubator, which is an experiment support system. Cells were fixed by injecting them with fixative at different time intervals. In addition to stationary experiment slots, Simbox provides a 1 g reference centrifuge. Cell fixation units were mounted in microgravity and 1 g reference positions of Simbox. Two Simbox incubators were used, one for space flight and the other as ground reference. Cells were fixed soon after launch and shortly before return of the spaceship. Due to technical problems, only early in-flight samples (about 40 min after launch microgravity and corresponding 1 g reference) were fully mixed with fixative, therefore only data from those samples are presented. Transcription of several genes involved in signal transduction, oxidative stress defence, cell cycle regulation and heat shock responses was investigated with quantitative PCR. The data indicate that Euglena cells suffer stress upon short-term exposure to microgravity; various stress-induced genes were up-regulated. Of 32 tested genes, 18 were up-regulated, one down-regulated and the rest remained unaltered. These findings are in a good agreement with results from other research groups using other organisms.

The effects of temperature and ultraviolet radiation (UVR; 280-400 nm) on seasonal succession in phytoplankton assemblages of Patagonia (Argentina) were studied in the context of global change. Samples collected during pre-bloom, bloom onset, bloom, and spring were exposed to in situ and increased (+4uC) temperatures and solar radiation with and without UVR. Daily cycles of effective photochemical quantum yield exhibited a pattern of high values in the morning, decreasing towards noon, and increasing in the afternoon. The decrease in yields towards noon as the season progressed increased from 30% in the pre-bloom to 80% in the spring; in the latter there were significant differences between radiation treatments under both temperature conditions. The highest inhibition rates were during the bloom, whereas the highest recovery rates were during the spring. Inhibition rates were generally higher in treatments exposed to UVR in comparison to photosynthetically active radiation-only treatments and some stages of the succession exhibited an additional temperature effect. Increasing temperatures had little effect on pre-bloom communities but helped to counteract the magnitude of the yield decrease during the bloom onset. However, during the bloom and in the spring, temperature and UVR acted synergistically, increasing the overall photochemical inhibition. Feedback mechanisms of increased temperatures causing a shallower mixing depth will expose phytoplankton to higher radiation, which will have a negative effect on the bloom and on spring assemblages. Due to the differential effects of solar UVR and increased temperature on phytoplankton, future studies should consider the repercussions on higher trophic levels. Fil: Villafañe, Virginia Estela. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Banaszak, Anastazia T.. Universidad Nacional Autónoma de México; México Fil: Guendulain García, Sergio D.. Universidad Nacional Autónoma de México; México Fil: Strauch, Sebastian M.. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina Fil: Halac, Silvana R.. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Secretaria de Ambiente y Desarr.sustentable de la Nacion. Instituto Nacional del Agua y del Ambiente(cordoba); Argentina Fil: Helbling, Eduardo Walter. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

During the late austral spring of 2009 we carried out experiments (4 days of duration) with four cyanobacteria species, Anabaena sp., Nostoc sp., Arthrospira platensis and Microcystis sp., to assess the combined effects of temperature and solar radiation on photosynthesis performance and morphology. Two experimental temperatures (18 °C and 23 °C, simulating a 5 °C increase under a scenario of climate change) and three radiation treatments (by using different filters/materials) were implemented: (i) P (PAR, 400–700 nm), (ii) PA (PAR + UV-A, 320–700 nm) and, (iii) PAB (PAR + UV-A + UV-B, 280–700 nm). In general, samples under the P treatment had less decrease/higher recovery rates of effective photochemical quantum yield (Y) than those receiving UV-A or UV-A + UV-B. The effects of increased temperature were species-specific: At the end of the experiments, it was seen that increased temperature benefited photosynthetic performance of Anabaena sp. and Nostoc sp. but not of Microcystis sp. and A. platensis. Higher temperature was also associated to an increase in the chain area of Anabaena sp., and to bigger trichomes in A. platensis; however, no morphological effects were observed in Microcystis sp. In addition, in Nostoc sp. the increase in temperature counteracted the UVR impact on the reduction of the chain area. How these effects and mechanisms will affect the trophodynamics and production of aquatic ecosystems is still uncertain, but the specificity of the responses suggests that not all cyanobacteria would be equally benefited by temperature increases therefore affecting the balance and interaction among species in the water column.