Graviperception and gravitaxis in algae.

Photosynthetic flagellates are among the most intensely studied unicellular organisms in the field of graviperception and gravitaxis. While the phenomenon of graviorientation has been known for many decades, only recently was the molecular mechanism unveiled. Earlier hypotheses tried to explain the precise orientation by a passive buoy mechanism assuming the tail end to be heavier than the front. In the photosynthetic flagellate Euglena gracilis, the whole cell body is denser than the surrounding medium, pressing onto the lower cell membrane where it seems to activate mechanosensitive ion channels specific for calcium. The calcium entering the cells during reorientation can be visualized by the fluorescence probe, Calcium Crimson. Cyclic AMP is likewise involved in the molecular pathway. Inhibitors of calcium channels and ionophores impair gravitaxis while caffeine, a blocker of the phosphodiesterase, enhances the precision of orientation.     

Physiological characterization of gravitaxis in Euglena gracilis and Astasia longa studied on sounding rocket flights.

Euglena gracilis is a photosynthetic, unicellular flagellate found in eutrophic freshwater habitats. The organisms control their vertical position in the water column using gravi- and phototaxis. Recent experiments demonstrated that negative gravitaxis cannot be explained by passive buoyancy but by an active physiological mechanism. During space experiments, the threshold of gravitaxis was determined to be between 0.08 and 0.12 x g. A strong correlation between the applied acceleration and the intracellular cAMP and Ca2+ was observed. The results support the hypothesis, that the cell body of Euglena, which is denser than the surrounding medium exerts a pressure onto the lower membrane and activates mechanosensitive Ca2+ channels. Changes in the membrane potential and the cAMP concentration are most likely subsequent elements in a signal transduction chain, which results in reorientation strokes of the flagellum.     

Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: Involvement of a transient receptor potential-like channel and a calmodulin

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavior responses. The organism shows pronounced negative gravitaxis. This movement is based on physiological mechanisms, which in the past had been only indirectly assessed. It was shown that mechano-sensitive calcium channels are involved in the gravitaxis response. Recent studies have demonstrated that members of the transient receptor potential (TRP) family function as mechano-sensitive channels in several different cell types. We have sequenced part of a TRP gene in Euglena and applied RNA interference (RNAi) to confirm that these channels are involved in graviperception. It was found that RNAi against the putative TRP channel abolished gravitaxis. The genes of three calmodulins were sequences in Euglena, one of which was previously known in its protein structure (cal 1). The other two were unknown (cal 2 and cal 3). Cal 2 has been analyzed in detail. The biosynthesis of the corresponding proteins of cal 1 and cal 2 was inhibited by means of RNA interference to see whether this blockage impairs gravitaxis. RNAi of cal 1 leads to a long-term loss of free swimming in the cells (while euglenoid movement persists). It induced pronounced cell form aberrations and the division of cells was hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Thus cal 1 does not seem to be involved in gravitaxis. In contrast, the blockage of cal 2 has no pronounced influence on motility and cell form but leads to a complete loss of gravitactic orientation for more than 30 days showing that this calmodulin is an element in the signal transduction chain. The data are discussed in the context of the current model of the gravitaxis signal transduction chain in Euglena gracilis.     

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.     

Experimental and mathematical model of the interactions in the mixed culture of links in the “producer–consumer” cycle

The paper presents a experimental and mathematical model of interactions between invertebrates (the ciliates Paramecium caudatum and the rotifers Brachionus plicatilis) in the “producer–consumer” aquatic biotic cycle with spatially separated components. The model describes the dynamics of the mixed culture of ciliates and rotifers in the “consumer” component, feeding on the mixed algal culture of the “producer” component. It has been found that metabolites of the algae Scenedesmus produce an adverse effect on the reproduction of the ciliates P. caudatum. Taking into account this effect, the results of investigation of the mathematical model were in qualitative agreement with the experimental results. In the “producer–consumer” biotic cycle it was shown that coexistence is impossible in the mixed culture of invertebrates of the “consumer” component. The ciliates P. caudatum are driven out by the rotifers B. plicatilis.     

Gravitaxis and graviperception in flagellates.

There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column. In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS). During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years. Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion. Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane. This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis. The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane. Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR. The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification. This result was confirmed during recent parabolic flights. The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum. cAMP plays a role as a secondary messenger. Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen. Preliminary experiments are discussed.     

Gravitaxis and graviperception in Euglena gracilis.

Gravitactic orientation in the flagellate Euglena gracilis is mediated by an active physiological receptor rather than a passive alignment of the cells. During a recent space flight on the American shuttle Columbia the cells were subjected to different accelerations between 0 and 1.5 x g and tracked by computerized real-time image analysis. The dependence of orientation on acceleration followed a sigmoidal curve with a threshold at < or = 0.16 x g and a saturation at about 0.32 x g. No adaptation of the cells to the conditions of weightlessness was observed over the duration of the space mission (12 days). Under terrestrial conditions graviorientation was eliminated when the cells were suspended in a medium the density of which (Ficoll) equaled that of the cell body (1.04 g/ml) and was reversed at higher densities indicating that the whole cytoplasm exerts a pressure on the respective lower membrane. There it probably activates stretch-sensitive calcium specific ion channels since gravitaxis can be affected by gadolinium which is a specific inhibitor of calcium transport in these structures. The sensory transduction chain could involve modulation of the membrane potential since ion channel blockers, ionophores and ATPase inhibitors impair graviperception.     

Comparative studies of the graviresponses of Paramecium and Loxodes.

Gravitactic protozoa offer advantages in studying how the gravity stimulus is perceived on the cellular level. By means of a slow rotating centrifuge microscope in space the acceleration thresholds for gravitaxis of Loxodes striatus and Paramecium biaurelia were determined: < or = 0.15 x g for Loxodes and 0.3 x g for Paramecium, indicating different sensitivities of these species. Neutral-buoyant densities of immobilized cells were determined using media of different densities, revealing densities of 1.03 to 1.035 g/cm3 for Loxodes and 1.04 g/cm3 to 1.045 g/cm3 for Paramecium. Behavioral studies revealed that gravitaxis of Loxodes persisted independent of the density of the medium. In contrast, negative gravitaxis of Paramecium was no longer measurable if the density of the medium approached the density of the cell. The results suggest that in the case of Loxodes gravity is perceived by an intracellular receptor and, in the case of Paramecium by its own mass via the pressure on the lower cell membrane.     

How gravity acts on Paramecium: new insights from free-fall experiments.

The swimming behaviour of Paramecium is affected by media of various specific gravities. At 1g, the negative gravitaxis of Paramecium virtually disappears in solutions the specific gravity of which is about the same as that of the organism (1.04). In solutions with a higher specific gravity (1.08), Paramecium becomes positively gravitactic. We recorded the swimming tracks of Paramecium in these media on videotape before, during and after free-falls. The records show that the density-dependent differences in the swimming behaviour disappeared immediately following the onset of the free-fall. The recorded tracks and distributions of cells in the experimental chambers were compared with computer-simulated traces and distributions based on gravitactic and gravikinetic models proposed for Paramecium. Our preliminary analysis favors a novel gravitactic mechanism involving modification of the ciliary movement The drop shaft at the Japan Microgravity Center, Hokkaido (JAMIC) was used for the free-fall experiments.     

A theory of gravikinesis in Paramecium.

The archaic eukaryote unicellular microorganism, Paramecium, is propelled by thousands of cilia, which are regulated by modulation of the membrane potential. Ciliates can successfully cope with gravity, which is the phylogenetically oldest stimulus for living things. One mechanism for overcoming sedimentation is negative gravitaxis, an orientational response antiparallel to the gravity vector. We have postulated the existence of a negative gravikinesis in Paramecium, i.e. a modulation of swimming speed as a function of cellular orientation in space. With negative gravikinesis, an upward oriented cell actively augments the rate of forward swimming and depresses active locomotion during downward orientation. A brief outline of the gravikinesis hypothesis is given on a quantitative basis and experimental data are presented which have confirmed the major assumptions.     

Effects of gamma-ray and high energy carbon ion irradiation on swimming velocity of Euglena gracilis.

The effects of gamma-ray and high energy carbon ion irradiation on the swimming velocity of the photosynthetic flagellate Euglena gracilis strain Z were studied, focusing on a dose-effect relationship. Cells were exposed to 60Co gamma-rays at 6 doses of 10, 15, 20, 40, 100 and 200 Gy for water, and also to 290 MeV/amu carbon ions from the Heavy Ion Medical Accelerator in Chiba at 7 doses (5, 10, 15, 20, 50, 100 and 200 Gy for water). The swimming velocity was measured by a biomonitoring system, called ECOTOX. The swimming velocities of Euglena gracilis cells were significantly decreased by >40 Gy gamma-rays and >5 Gy carbon ions, respectively. The 50% effective doses for inhibition, 34 +/- 4 Gy (gamma-rays) and 13 +/- 1 Gy (290 MeV/amu carbon ions), were estimated from the best fit to data of the logistic model. The relative biological effectiveness (2.6 +/- 0.4) was calculated by the ratio of 50% effective doses. The inhibition of the swimming velocity of the cells irradiated with gamma-rays was still present after 3 days, while recovery of the swimming velocity was shown in the cells exposed to 290 MeV/amu carbon ions. It is suggested that ionizing radiation inhibits ATP production and/or increases frictional drag on beating of the flagellum, thus decreasing swimming velocity.     

Protozoa as model systems for the study of cellular responses to altered gravity conditions.

The orientation behavior of Paramecium changed in a similar way after transition to conditions of free-fall in a sounding rocket and after transition to conditions of simulated weightlessness on a fast rotating clinostat. After a period of residual orientation, Paramecium cells distributed themselves randomly 80 s (120 s) after onset of free-fall (simulated weightlessness). Swimming velocity increased significantly; however, the increase was transient and subsided after 3 min in the rocket experiments, while the velocity remained enhanced even during 2 h of rotation on a fast clinostat. Trichocysts were present and without morphological changes in Paramecium cells which had been exposed to a rocket flight, as well as to fast or slow rotation on a clinostat. Regeneration of the oral apparatus of Stentor and morphogenesis of Eufolliculina proceeded normally on the clinostat. The results demonstrate that the clinostat is a useful tool to simulate the conditions of weightlessness on earth and to detect gravisensitive cellular functions.     

Competition between links in "producer-consumer" trophic chains in an aquatic closed system with spatially separated components.

The work analyzes functioning of a "producer-consumer" closed aquatic system with spatially separated links, where each component consisted of two species. Producers in the system were the microalgae of Chlorella vulgaris and Scenedesmus sp., consumers--Paramecium caudatum infusoria and Brachionus sp. rotifers. In the experiment the competing predators were consuming on a mixed culture of algae, and the competition of algae was studied under nitrogen limitation. Under these conditions competitiveness of Scenedesmus was higher than that of Chlorella vulgaris. Metabolism products of Scenedesmus algae have been found to have negative effect on reproduction of Paramecium caudatum protozoa. Predator population dynamics in the "consumer" link demonstrated that the rotifers that consume two algal species are more competitive compared to protozoa feeding on chlorella only. Grant numbers: N99-04-96017, N25.     

Putative graviperception mechanisms of protists.

Many (if not all) free-living cells use the gravity vector for their spatial orientation (gravitaxis). Additional responses may include gravikinesis as well as changes in morphological and physiological parameters. Though using essentially different modes of locomotion, ameboid and ciliated cells seem to rely on common fundamental graviperception mechanisms. Uniquely in the ciliate family Loxodidae a specialized intracellular gravireceptor organelle has been developed, whereas in all other cells common cell structures seem to be responsible for gravisensing. Changes in direction or magnitude of acceleration (from 0 to 5 g) as well as experiments in density-adjusted media strongly indicate that either the whole cytoplasm or dense organelles like nuclei act as statoliths and open directly or via cytoskeletal elements mechano-sensitive ion channels in the cell membrane. A recent spaceflight experiment (S/MM-06) demonstrated that prolonged (9 d) actual weightlessness did not affect the ability of Loxodes to respond to acceleration stimuli. However, prolonged cooling (> or = l4 d, 4-10 degrees C) destroyed the ability for gravitactic orientation of Paramecium. This may reflect a profound effect either on the gravireceptor itself or on the gravity-signal processing. In gravity signalling the ubiquitous second messenger cAMP may be involved in acceleration-stimulus transduction.     

Paramecium--a model system for studying cellular graviperception.

Experiments under varied gravitational accelerations as well as in density-adjusted media showed that sensation of gravity in protists may be linked to the known principles of mechanosensation. Paramecium, a ciliate with clear graviresponses (gravitaxis and gravikinesis) is an ideal model system to prove this hypothesis since the ciliary activity and thus the swimming behaviour is controlled by the membrane potential. It has also been assumed that the cytoplasmic mass causes a distinct stimulation of the bipolarly distributed mechano-sensitive K+ and Ca2+ ion channels in the plasma membrane in dependence of the spatial orientation of the cell. In order to prove this hypothesis, different channel blockers are currently under investigation. Gadolinium did not inhibit gravitaxis in Paramecium, showing that it does not specifically block gravireceptors. Further studies concentrated on the question of whether second messengers are involved in the gravity signal transduction chain. Exposure to 5 g for up to 10 min led to a significant increase in cAMP.     

Competition of the natural and manmade biotic cycles in the closed aquatic system.

This study addresses competition between the Paramecium bursaria and zoochlorella-endosymbiosis and the infusoria Paramecium caudatum in a closed aquatic system. The system is a natural model of a simple biotic cycle. P. bursaria consumes glucose and oxygen released by its zoochlorella and releases nitrogenous compounds and carbon dioxide necessary for algal photosynthesis. P. caudatum was fed on bacteria. It was shown that the infusoria P. bursaria united in one cycle with Chlorella had a higher competitive ability than P. caudatum. With any initial percentage of the infusoria in the mixed culture, the end portion of P. bursaria reached 90-99%, which was significantly higher than the end potion of the P. caudatum population. It is assumed that the sustenance expenditures of P. caudatum were greater than those of the endosymbiotic paramecium, i.e. the closing of the components into a biotic cycle leads to a decrease in sustenance expenditures.     

Electric potentiation of gravikinesis in Paramecium is possibly mediated by filaments.

Sensitivity of Paramecium to mechanical stress including gravitational force is organized along two opposing gradients of membrane channel distribution: depolarizing Ca channels and hyperpolarizing K channels. Mechanoreceptor channels reside in the membrane of the cell soma and are activated, when the weight of the cytoplasm deforms the "lower" plasma membrane. Channel distribution is such as to generate ciliary activation which can counteract sedimentation of the cells: a reduction in downward swimming rate and an augmentation in upward swimming rate. Application of weak DC fields does not only induce the well-known cathodal orientation and swimming of Paramecium toward the cathode (galvano-taxis). We document that swimming velocity is augmented up to 175% as a function of the voltage gradient between 0.3 V/cm and 0.8 V/cm (galvanokinesis). A gradient of 0.3 V/cm was highly effective in raising the common negative gravikinesis of downward swimmers threefold. The gravikinesis of upward swimmers reversed polarity under field stimulation inducing cells to augment sedimentation effects (positive gravikinesis). Both effects of electric-field stimulation on ciliary activation are of the depolarizing type: reduction in the frequency of normally beating cilia. Analysis of the data shows that a voltage-sensitivity of gravireceptor channels would not account for the observed potentiation of negative gravikinesis. It is suggested that a previously described voltage-dependent Ca channel of the soma membrane interferes with a Ca(2+)-sensitive, peripheral filament system, which directly connects to gravireceptor channels.     

The magnetic fields of Mercury,Venus and Mars

Just as clearly as Mariner 10 established that Mercury has an intrinsic magnetic field, the Pioneer Venus orbiter has established that Venus has no significant intrinsic field. This is perhaps the opposite of what might be expected. Mercury, a small planet might be expected to cool rapidly and its internal dynamo to cease, while Venus, which is almost as large as the Earth, should not have lost much heat. On the contrary the source of energy of the Mercury dynamo appears to be extant whereas that of Venus appears to be extinct.The existence of a Martian magnetic field is controversial. No unambiguous signature of a Martian magnetic field has been reported. If the field on the nightside of Mars is of planetary rather than solar origin the Russian Mars spacecraft observations indicate the Martian dipole lies near the planetary equator rather than its pole.     

Graviperception in ciliates: steps in the transduction chain.

Ciliates represent suitable model systems to study the mechanisms of graviperception and signal transduction as they show clear gravity-induced behavioural responses (gravitaxis and gravikinesis). The cytoplasm seems to act as a "statolith" stimulating mechanosensitive ion channels in the cell membrane. In order to test this hypothesis, electrophysiological studies with Stylonychia mytilus were performed, revealing the proposed changes (de- or hyperpolarization) depending on the cell's spatial orientation. The behaviour of Paramecium and Stylonychia was also analyzed during variable acceleration conditions of parabolic flights (5th German Parabolic Flight Campaign, 2003). The corresponding data confirm the relaxation of the graviresponses in microgravity as well as the existence of thresholds of graviresponses, which are found to be in the range of 0.4xg (gravikinesis) and 0.6xg (gravitaxis).