The pleurodele, an animal model for space biology studies.

Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in vivo fertilization and development ("FERTILE" project); 2) influence of microgravity and space radiation on the organization and preservation of specialized structures in the neurons and muscle cells (in vitro; "CELIMENE" PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).     

Regulative development of Xenopus laevis in microgravity.

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.     

Influence of long-term altered gravity on the swimming performance of developing cichlid fish: including results from the 2nd German Spacelab Mission D-2.

This study presents qualitative and quantitative data concerning gravity-dependent changes in the swimming behaviour of developing cichlid fish larvae (Oreochromis mossambicus) after a 9 resp. 10 days exposure to increased acceleration (centrifuge experiments), to reduced gravity (fast-rotating clinostat), changed accelerations (parabolic aircraft flights) and to near weightlessness (2nd German Spacelab Mission D-2). Changes of gravity initially cause disturbances of the swimming performance of the fish larvae. With prolonged stay in orbit a step by step normalisation of the swimming behaviour took place in the fish. After return to 1g earth conditions no somersaulting or looping could be detected concerning the fish, but still slow and disorientated movements as compared to controls occurred. The fish larvae adapted to earth gravity within 3-5 days. Fish seem to be in a distinct early developmental stages extreme sensitive and adaptable to altered gravity; However, elder fish either do not react or show compensatory behaviour e.g. escape reactions.     

Changes in symbiotic and associative interrelations in a higher plant-bacterial system during space flight.

The miniature cenosis consisting of the water fern Azolla with its associated symbiotic nitrogen-fixing cyanobacterium Anabaena and the concomitant bacteria was investigated. Ecological closure was shown to produce sharp quantitative and qualitative changes in the number and type of concomitant bacteria. Changes in the distribution of bacterial types grown on beef-extract broth after space flight were recorded. Anabaena azollae underwent the most significant changes under spaceflight conditions. Its cell number per Azolla biomass unit increased substantially. Thus closure of cenosis resulted in a weakening of control over microbial development by Azolla. This tendency was augmented by spaceflight factors. Reduction in control exerted by macro-organisms over development of associated micro-organisms must be taken into account in constructing closed ecological systems in the state of weightlessness.     

The reaction of Xenopus laevis Daudin (South African toad) to linear accelerations.

Preparing the German Spacelab Mission D-2 project "Gravity Perception and Neuronal Plasticity"--STATEX II--ground based experiments have been performed with larvae of the amphibian vertebrate Xenopus laevis Daud. to study the reactions to different levels of acceleration forces and profiles. The larvae have been exposed to accelerations of up to 5 g for different time periods using a modified laboratory centrifuge and the NIZEMI (Niedergeschwindigkeits-Zentrifugen-Mikroskop) which allows direct observation and video documentation. The results will be discussed and compared with those of the D1-Mission, parabolic flights, and simulated weightlessness.     

Light microscopic analysis of the gravireceptor in Xenopus larvae developed in hypogravity.

The paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.     

Evidence that far-infrared radiation promotes growth of Xenopus laevis.

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.     

Evidence that far-infrared radiation promotes growth of Xenopus laevis

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.     

Neuroplastic reactivity of fish induced by altered gravity conditions: a review of recent results.

A review is being presented concerning behavioural, biochemical, histochemical and electronmicroscopical data on the influence of altered gravitational forces on the swimming performance and on the neuronal differentiation of the brain of cichlid fish larvae and adult swordtail fish that had been exposed to hyper-gravity (3g in laboratory centrifuges), hypo-gravity (>10(-2) g in a fast-rotating clinostat) and to near weightlessness (10(-4) g aboard the Spacelab D-2 mission). After long-term alterations of gravity (and parallel light deprivation), initial disturbances in the swimming behaviour followed by a stepwise regain of normal swimming modes are induced. Parallel, neuroplastic reactivities on different levels of investigation were found, such as adaptive alterations of activities of various enzymes in whole brain as well as in specific neuronal integration centers and an intraneuronal reactivity on ultrastructural level in individual brain parts and in the sensory epithelia of the inner ear. Taken together, these data reveal distinct adaptive neuroplastic reactions of fish to altered gravity conditions.     

Behavioural and biochemical investigations of the influence of altered gravity on the CNS of aquatic vertebrates during ontogeny.

Quantitative data are presented on the influences of hyper-gravity (3 +/- 1g) and of simulated weightlessness (approximately 0g) during early ontogeny of cichlid fish (Oreochromis mossambicus) and clawed toad (Xenopus laevis, Daudin) demonstrating changes in the swimming behaviour and the brain energy and plasma membrane metabolism. After return to 1g conditions, hyper-g reared fish and toads express the well known "loop-swimming" behaviour. By means of a computer based video analyzing system different types of swimming movements and velocities were quantitatively determined. Analyses of the brain energy and plasma-membrane metabolism of hyper-g fish larvae demonstrated an increase in energy availability (glucose 6Pi dehydrogenase, G-6P-DH), a decrease of cellular energy transformation (creatine kinase activity, CK) but no changes in energy consumptive processes (e.g. ATPases) and cytochrome oxidase activity (Cyt.-Ox). In contrast hypo-g fish larvae showed a slight increase in brain CK activity. In addition, unlike 1g controls, hyper-g fish larvae showed pronounced variations in the composition (=polarity) of sialoglycosphingolipids (=gangliosides), typical constituents of the nerve cell membranes, and a slight increase in the activity of sialidase, the enzyme responsible for ganglioside degradation.     

Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in urodeles.

Two species of newts (Urodela) and two types of clinostats for fast clinorotation (60 rpm) were used to investigate the influence of simulated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, differentiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with Triturus vulgaris using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles waltl using both horizontal and radial clinostats the results were contradictory compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictory results.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

Aquatic animals have almost no body weight related proprioception for spatial orientation. larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity.

For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission).

The further evaluation of the “weightless-larvae” revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls.

An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown eggs.

The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.     

Performance of a simple Closed Aquatic Ecosystem (CAES) in space.

A simple Closed Aquatic Ecosystem (CAES) consisting of single-celled green algae (Chlorella pyrenoidosa, producer), a spiral snail (Bulinus australianus, consumer) and a data acquisition and control unit was flown on the Chinese Spacecraft SHENZHOU-II in January 2001 for 7 days. In order to study the effect of microgravity on the operation of CAES, a 1 g centrifuge reference group in space, a ground 1 g reference group and a ground 1 g centrifuge reference group (1.4 g group) were run concurrently. Real-time data about algae biomass (calculated from transmission light intensity), temperature, light and centrifugation of the CAES were logged at minute intervals. It was found that algae biomass of both the microgravity group and the ground 1 g-centrifuge reference group (1.4 g) fluctuated during the experiment, but the algae biomass of the 1 g centrifuge reference group in space and the ground 1 g reference group increased during the experiment. The results may be attributable to influences of microgravity and 1.4 g gravity on the algae and snails metabolisms. Microgravity is the main factor to affect the operation of CAES in space and the contribution of microgravity to the effect was also estimated. These data may be valuable for the establishment of a complex CELSS in the future.     

Differentiation in microgravity of neural and muscle cells of a vertebrate (amphibian).

The CELIMENE space experiment (CELulles en Impesanteur: Muscle Et Neurone Embryonnaires) was devoted to the study of the influence of gravity on the differentiation, the organisation and the maintenance of the highly specialised nervous system and muscular system. CELIMENE was carried out during the first flight of the IBIS hardware (Instrument for BIology in Space) with the fully automatic space mission PHOTON 10 in February 1995. Using the amphibian Pleurodeles waltl as a vertebrate model, in vitro experiments involved immunocytochemical detection of glial-, neuronal- and muscle-specific markers, and neurotransmitters in cells developed under conditions of microgravity compared with 1g controls, on-board and on the ground. We observed that the altered gravity did not disturb cell morphogenesis or differentiation.     

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.     

Design of specific hardware to obtain embryos and maintain adult urodele amphibians aboard a space station.

The study of the influence of weightlessness on fertilization and embryonic development of a vertebrate is of importance in the understanding of basic embryogenesis and in the preparation of the future exploration of space. Accordingly, specific hardware was designed to perform experiments on board the MIR space station with an amphibian vertebrate model, taking into account the biological requirements and the multiple constraints of a long-term mission. This paper describes the biological uses and presents the technological specifications of the device developed under CNES management. The hardware was adapted to and is compatible with biological requirements as confirmed by three experiments performed in space on board the orbital MIR station.     

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) "morphological patterning" such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) "the timing of embryonic events" such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.     

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.     

In vivo fertilization and development in microgravity using pleurodele ("ZEUS" project).

The objectives of this experiment are to perform natural fertilization and to achieve embryonic development in microgravity. Pleurodeles waltl, an urodele amphibian, is considered by CNES and NASA to be suitable experimental material for achieving in vivo fertilization in space. Previously inseminated females can be embarked in the Frog Environmental Unit (FEU) developed by NASA. Laying of eggs will be provoked by hormonal stimulation in flight and development will be followed. Various technical problems have been resolved in laboratory experiments and during parabolic flights : the time of hormone stimulation after insemination, choice of hormone guaranteeing [correction of guarenteing] 95% success, other factors conditioning [correction of conditionning] the laying, experimental procedures to study developmental kinetics at phenotypic levels, and selection of cellular and molecular markers of development.