Changes in Gravitaxis and Gene-Expression in an Euglena gracilis Culture over Time.

Age-dependent changes in the transcription levels of 5-day-old Euglena gracilis cells, which showed positive gravitaxis, 6-day-old cells without gravitactic orientation, and older cells (9- and 11-day-old, which displayed a precise negative gravitaxis) were determined through microarray analysis. Hierarchical clustering of four independent cell cultures revealed pronounced similarities in transcription levels at the same culture age, which proves the reproducibility of the cultivation method. Employing the non-oriented cells from the 6-day-old culture as a reference, about 2779 transcripts were found to be differentially expressed. While positively gravitactic cells (5-day-old culture) showed only minor differences in gene expression compared to the 6-day reference, pronounced changes of mRNAs (mainly an increase) were found in older cells compared to the reference culture. Among others, genes coding for adenylyl cyclases, photosynthesis, and metabolic enzymes were identified to be differentially expressed. The investigated cells were grown in batch cultures, so variations in transcription levels most likely account for factors such as nutrient depletion in the medium and self-shading. Based on these findings, a particular transcript (e.g., transcript 19556) was downregulated using the RNA interference technique. Gravitaxis and phototaxis were impaired in the transformants, indicating the role of this transcript in signal transduction. Results of the experiment are discussed regarding the increasing importance of E. gracilis in biotechnology as a source of valuable products and the possible application of E. gracilis in life-support systems.

Snakes on a slope: strong anti-gravitactic responses and differential habitat use in the Saharan horned viper (Cerastes cerastes) in the Negev desert.

The way species use their habitat dictates their intra- and interspecific interactions. We studied the effects of the microhabitat type and slope on the movement behaviour of the Saharan horned viper (Cerastes cerastes) in its natural habitat. This viper occurs in sand dunes and moves mostly by sidewinding. Additionally, we studied the microhabitat preference of desert rodents-the vipers' main prey. We placed the vipers on different natural dune slopes and recorded their behaviour. We found a strong anti-gravitactic response: vipers moved more frequently towards the top of the dune than in any other direction, despite a decrease in stride length with increasing slope. The foraging-related behaviour of the vipers was concentrated in the dune semi-stable areas rather than its stable or shifting sand areas. We measured rodent activity by placing seed trays in the dune allowing the rodents to collect seeds. Rodent activity was the highest in the shifting sands, closely followed by the semi-stable microhabitat. These results suggest the vipers use the semi-stable microhabitat mainly for foraging and may use the shifting sand areas as commuting routes between such areas. This study may be of use for conservation efforts of psammophilic species in desert dunes.

Gravitactic Swimming of the Planula Larva of the Coral Acropora: Characterization of Straightforward Vertical Swimming.

Vertical migration as well as horizontal dispersion is important in the ecological strategy of planktonic larvae of sedentary corals. We report in this paper unique vertical swimming behavior of planulae of the reef-building coral Acropora tenuis. Several days after fertilization, most of the planulae stayed exclusively at either the top or the bottom of the rearing tank. A good proportion of the planulae migrated almost vertically between top and bottom with fairly straight trajectories. Planulae sometimes switched their swimming direction via a sharp turn between the opposite directions. Quantitative analyses demonstrated that planulae kept constant speed while swimming either upward or downward, in contrast to frequent changes of direction and speed in horizontal swimming. Statistical comparison of propulsive speeds, estimated from swimming speeds and passive sedimentation, revealed gravikinesis of planulae, where the propulsive speed was significantly greater in downward swimming than upward swimming. The larval density hydrodynamically estimated was 0.25% lower than sea water density, which might be explained by the large quantity of lipids in planulae. Also, the deciliated larvae tended to orient oral end-up during floatation, presumably due to asymmetrical distribution of the endogenous light lipids. Plasticity of the larval tissue geometry could easily cause relocation of the center of forces which work together to generate gravitactic-orientation torque and, therefore, abrupt changing of the gravitactic swimming direction. The bimodal gravitactic behavior may give a new insight into dispersal and recruitment of coral larvae.

3D escape: an alternative paradigm for spatial orientation studies in insects.

Arthropods and in particular insects show a great variety of different exoskeletal sensors. For most arthropods, spatial orientation and gravity perception is not fully understood. In particular, the interaction of the different sensors is still a subject of ongoing research. A disadvantage of most of the experimental methods used to date to study the spatial orientation of arthropods in behavioral experiments is that the body or individual body parts are fixed partly in a non-natural manner. Therefore, often only the movement of individual body segments can be used to evaluate the experiments. We here present a novel experimental method to easily study 3D-escape movements in insects and analyze whole-body reaction. The animals are placed in a transparent container, filled with a lightweight substrate and rotating around two axes. To verify our setup, house crickets (Acheta domesticus) with selectively manipulated gravity-perceiving structures were analyzed. The spatial orientation behavior was quantified by measuring the time individuals took to escape toward the surface and the angular deviation toward the gravitational vector. These experiments confirm earlier results and therefore validated our experimental setup. Our new approach thus allows to investigate several comprehensive questions regarding the spatial orientation of insects and other animals.

Euglena, a Gravitactic Flagellate of Multiple Usages.

Human exploration of space and other celestial bodies bears a multitude of challenges. The Earth-bound supply of material and food is restricted, and in situ resource utilisation (ISRU) is a prerequisite. Excellent candidates for delivering several services are unicellular algae, such as the space-approved flagellate Euglena gracilis. This review summarizes the main characteristics of this unicellular organism. Euglena has been exposed on various platforms that alter the impact of gravity to analyse its corresponding gravity-dependent physiological and molecular genetic responses. The sensory transduction chain of gravitaxis in E. gracilis has been identified. The molecular gravi-(mechano-)receptors are mechanosensory calcium channels (TRP channels). The inward gated calcium binds specifically to one of several calmodulins (CaM.2), which, in turn, activates an adenylyl cyclase. This enzyme uses ATP to produce cAMP, which induces protein kinase A, followed by the phosphorylation of a motor protein in the flagellum, initiating a course correction, and, finally, resulting in gravitaxis. During long space missions, a considerable amount of food, oxygen, and water has to be carried, and the exhaled carbon dioxide has to be removed. In this context, E. gracilis is an excellent candidate for biological life support systems, since it produces oxygen by photosynthesis, takes up carbon dioxide, and is even edible. Various species and mutants of Euglena are utilized as a producer of commercial food items, as well as a source of medicines, as it produces a number of vitamins, contains numerous trace elements, and synthesizes dietary proteins, lipids, and the reserve molecule paramylon. Euglena has anti-inflammatory, -oxidant, and -obesity properties.

Molecular Cross-Talk between Gravity- and Light-Sensing Mechanisms in Euglena gracilis.

Euglena gracilis is a photosynthetic flagellate. To acquire a suitable position in its surrounding aquatic environment, it exploits light and gravity primarily as environmental cues. Several physiological studies have indicated a fine-tuned relationship between gravity sensing (gravitaxis) and light sensing in E. gracilis. However, the underlying molecular mechanism is largely unknown. The photoreceptor photoactivated adenylyl cyclase (PAC) has been studied for over a decade. Nevertheless, no direct/indirect interaction partner (upstream/downstream) has been reported for PAC. It has been shown that a specific protein, kinase A (PKA), showed to be involved in phototaxis and gravitaxis. The current study reports the localization of the specific PKA and its relationship with PAC.

The enigmatic Placozoa part 2: Exploring evolutionary controversies and promising questions on earth and in space.

The placozoan Trichoplax adhaerens has been bridging gaps between research disciplines like no other animal. As outlined in part 1, placozoans have been subject of hot evolutionary debates and placozoans have challenged some fundamental evolutionary concepts. Here in part 2 we discuss the exceptional genetics of the phylum Placozoa and point out some challenging model system applications for the best known species, Trichoplax adhaerens.

Caenorhabditis elegans exhibits positive gravitaxis.

BACKGROUND: Gravity plays an important role in most life forms on Earth. Yet, a complete molecular understanding of sensing and responding to gravity is lacking. While there are anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. Caenorhabditis elegans is suitable for gene discovery approaches that may help identify molecular mechanisms of gravity sensing. It is unknown whether C. elegans can sense the direction of gravity. RESULTS: In aqueous solutions, motile C. elegans nematodes align their swimming direction with the gravity vector direction while immobile worms do not. The worms orient downward regardless of whether they are suspended in a solution less dense (downward sedimentation) or denser (upward sedimentation) than themselves. Gravitaxis is minimally affected by the animals' gait but requires sensory cilia and dopamine neurotransmission, as well as motility; it does not require genes that function in the body touch response. CONCLUSIONS: Gravitaxis is not mediated by passive forces such as non-uniform mass distribution or hydrodynamic effects. Rather, it is mediated by active neural processes that involve sensory cilia and dopamine. C. elegans provides a genetically tractable system to study molecular and neural mechanisms of gravity sensing.

Temporal Evolution of the Gravitaxis of Euglena gracilis from a Single Cell.

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 mm × 2.5 mm × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized.

Locomotion and righting behavior of sea stars: a study case on the bat star Asterina stellifera (Asterinidae) / Comportamiento de locomoción y enderezamiento en estrellas de mar: un caso de estudio sobre la estrella Asterina stellifera (Asterinidae)

Introduction: The locomotion behavior of an organism involves the integration of aspects like body symmetry, sensory and locomotor systems. Furthermore, various ecological factors seem to be related to locomotion characteristics, such as foraging strategy, migration trends, response to predators and competitors, and environmental stress. Objective: To analyze locomotion and the influence of body symmetry in the crawling and righting movements of the sea star Asterina stellifera. Methods: We carried out laboratory experiments in aquariums in the presence/absence of water current and on a horizontal and vertical surface. Results: The speed is similar to speed in other species of similar size. Both the speed and linearity of displacement were independent of individual body size. A water current leads to faster crawling and straight paths, but there is no rheotaxis: streams do not affect locomotion. Speed and linearity of displacement were independent of individual body size. The displacement pattern described here may be an adaptation of organisms that present dense populations in communities with high prey abundance, as is the case of A. stellifera. Conclusions: Like other asteroids, this species did not show an Anterior/Posterior plane of symmetry during locomotion, or righting movement: it does not tend to bilaterality.

Introducción: El comportamiento de locomoción de un organismo implica la integración de aspectos como la simetría corporal, los sistemas sensorial y locomotor. Además, varios factores ecológicos parecen estar relacionados con las características de la locomoción, como la estrategia de alimentación, las tendencias migratorias, la respuesta a los depredadores y competidores y el estrés ambiental. Objetivo: Analizar el patrón general de locomoción y la influencia de la simetría corporal en la locomoción y enderezamiento de la estrella de mar Asterina stellifera. Métodos: Realizamos experimentos de laboratorio en acuarios en presencia / ausencia de corriente de agua y en superficie horizontal y vertical. Resultados: La velocidad es similar a la velocidad en otras especies de tamaño similar. Tanto la velocidad como la linealidad del desplazamiento fueron independientes del tamaño corporal individual. Una corriente de agua conduce a una velocidad de desplazamiento mayor y a trayectorias más rectas, pero no hay reotaxis: una corriente de agua no afecta el patrón de locomoción. La velocidad y la linealidad del desplazamiento fueron independientes del tamaño corporal individual. El patrón de desplazamiento aquí descrito puede ser una adaptación de organismos que presentan densas poblaciones en comunidades con alta abundancia de presas, como es el caso de A. stellifera. Conclusiones: Al igual que otros asteroides, esta especie no mostró un plano de simetría Anterior / Posterior durante la locomoción o el movimiento de enderezamiento: no tiende a la bilateralidad.

Antagonistic Inhibitory Circuits Integrate Visual and Gravitactic Behaviors.

Larvae of the tunicate Ciona intestinalis possess a central nervous system of 177 neurons. This simplicity has facilitated the generation of a complete synaptic connectome. As chordates and the closest relatives of vertebrates, tunicates promise insight into the organization and evolution of vertebrate nervous systems. Ciona larvae have several sensory systems, including the ocellus and otolith, which are sensitive to light and gravity, respectively. Here, we describe circuitry by which these two are integrated into a complex behavior: the rapid reorientation of the body followed by upward swimming in response to dimming. Significantly, the gravity response causes an orienting behavior consisting of curved swims in downward-facing larvae but only when triggered by dimming. In contrast, the majority of larvae facing upward do not respond to dimming with orientation swims-but instead swim directly upward. Under constant light conditions, the gravity circuit appears to be inoperable, and both upward and downward swims were observed. Using connectomic and neurotransmitter data, we propose a circuit model that can account for these behaviors. The otolith consists of a statocyst cell and projecting excitatory sensory neurons (antenna cells). Postsynaptic to the antenna cells are a group of inhibitory primary interneurons, the antenna relay neurons (antRNs), which then project asymmetrically to the right and left motor units, thereby mediating curved orientation swims. Also projecting to the antRNs are inhibitory photoreceptor relay interneurons. These interneurons appear to antagonize the otolith circuit until they themselves are inhibited by photoreceptors in response to dimming, thus providing a triggering circuit.

The shape effect of flagella is more important than bottom-heaviness on passive gravitactic orientation in Chlamydomonas reinhardtii.

The way the unicellular, biflagellated, green alga Chlamydomonas orients upward has long been discussed in terms of both mechanics and physiology. In this study, we focus on the mechanics, i.e. the 'passive' mechanisms, of gravitaxis. To rotate the body upwards, cellular asymmetry is critical. Chlamydomonas can be depicted as a nearly spherical cell body with two anterior, symmetric flagella. The present study looks at the question of whether the existence of the flagella significantly affects torque generation in upward reorientation. The 'density asymmetry model' assumes that the cell is spherical and bottom-heavy and that the shape and weight of the flagella are negligible, while the 'shape asymmetry model' considers the shape of the flagella. Both our experimental and simulation results revealed a considerable contribution from shape asymmetry to the upward orientation of Chlamydomonas reinhardtii, which was several times larger than that of density asymmetry. From the experimental results, we also quantified the extent of bottom-heaviness, i.e. the distance between the centers of gravity and the figure when the cell body is assumed to be spherical. Our estimation was approximately 30 nm, only one-third of previous assumptions. These findings indicate the importance of the viscous drag of the flagella to the upward orientation, and thus negative gravitaxis, in Chlamydomonas.

Responses of Bioconvection of Tetrahymena thermophila to Partial Gravity Created by Aircraft Flight.

Bioconvection is a form of collective pattern formation driven by negative gravitaxis of swimming microorganisms. In bioconvection, the interaction between individual swimmers results in self-organization leading to the development of a macroscopic structure typically 100-1000 times greater than an individual microorganism. To gain insight into the role of gravity in this self-organization phenomenon, we investigated the bioconvective behavior of the ciliate Tetrahymena thermophila under short-term partial gravity, i.e., gravitational acceleration < 1 g, achieved by quasiparabolic flight maneuvers of an aircraft. The bioconvective responses of T. thermophila were assessed by observing the collective motion simultaneously in two separate scales, which we call macroscale and microscale, using a newly designed "dual-objective" device with two different magnifications. Microscale analysis revealed that the magnitude of gravikinesis, i.e., active regulation of the propulsive thrust, decreased almost linearly with changes in gravitational acceleration, while gravitactic characteristics, assessed by the distribution of the swimming direction, did not change significantly during partial gravity. Macroscale analysis demonstrated that downward plumes of convection pattern gradually shortened from the lower end, and disappeared under partial gravity. The sustained time of the plumes decreased almost linearly with changes in gravitational acceleration. The response of downward plumes to partial gravity may be attributable to the accumulation of cells into blobs in downward migration, which increases the rate of downward migration enough to exceed the rate of upward movement, which is enhanced due to gravikinesis. This suggests that gravity may act on cells involved in collective pattern formation differently than on free-swimming cells.

Adaptive Changes in the Vestibular System of Land Snail to a 30-Day Spaceflight and Readaptation on Return to Earth.

The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

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.

Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii.

Drastic reorganization of the bioconvection pattern of Chlamydomonas: quantitative analysis of the pattern transition response.

Motile aquatic microorganisms are known to self-organize into bioconvection patterns. The swimming activity of a population of microorganisms leads to the emergence of macroscopic patterns of density under the influence of gravity. Although long-term development of the bioconvection pattern is important in order to elucidate the possible integration of physiological functions of individuals through bioconvection pattern formation, little quantitative investigation has been carried out. In the present paper, we present the first quantitative description of long-term behavior of bioconvection of Chlamydomonas reinhardtii, particularly focusing on the 'pattern transition response'. The pattern transition response is a sudden breakdown of the steady bioconvection pattern followed by re-formation of the pattern with a decreased wavelength. We found three phases in the pattern formation of the bioconvection of C. reinhardtii: onset, steady-state 1 (before the transition) and steady-state 2 (after the transition). In onset, the wavelength of the bioconvection pattern increases with increasing depth, but not in steady-states 1 or 2. By means of the newly developed two-axis view method, we revealed that the population of C. reinhardtii moves toward the bottom of the experimental chamber just before the pattern transition. This indicates that the pattern transition response could be caused by enhancement of the gyrotaxis of C. reinhardtii as a result of the changes in the balance between the gravitactic and gyrotactic torques. We also found that the bioconvection pattern changes in response to the intensity of red-light illumination, to which C. reinhardtii is phototactically insensitive. These facts suggest that the bioconvection pattern has a potential to drastically reorganize its convection structure in response to the physiological processes under the influence of environmental cues.