Vestibular and visual contribution to fish behavior under microgravity.

Vestibular and visual information are two major factors fish use for controlling their posture under 1 G conditions. Parabolic flight experiments were carried out to observe the fish behavior under microgravity for several different strains of Medaka fish (Oryzias latipes). There existed a clear strain-difference in the behavioral response of the fish under microgravity: Some strains looped, while other strains did not loop at all. However, even the latter strains looped under microgravity conditions when kept in complete darkness, suggesting the contribution of visual information to the posture control under microgravity. In the laboratory, eyesight (visual acuity) was checked for each strain, using a rotating striped-drum apparatus. The results also showed a strain-difference, which gave a clue to the different degree of adaptability to microgravity among different strains. Beside loopings, some fish exhibited rolling movement around their body axis. Tracing each fish during and between parabolas, it was shown that to which side each fish rolls was determined specifically to each individual fish, and not to each strain. Thus, rolling direction is not genetically determined. This may support the otolith asymmetry hypothesis. Fish of a mutant strain (ha strain, having homozygous recessive of one gene ha) have some malfunction in otolith-vestibular system, and their behavior showed they are not dependent on gravity. Morphological abnormalities of their ear vesicles during the embryonic and baby stages were noted. Their eyesight and dorsal light responses were also studied. Progress in the project of establishing a new strain which has good eyesight and, at the same time, being deficient in otolith-vestibular system was reported. Crosses between the strain of good eyesight and ha strain were made, and to some extent, F2 fish have already shown such characteristics suited for living under microgravity conditions.     

Use of an otolith-deficient mutant in studies of fish behavior in microgravity.

The mutant strain (ha) of medaka (Oryzias latipes) lack utricular otoliths as fry, and some never form otoliths for life. The cross (F1 generation) between the strain having good eyesight and another strain having ordinary eyesight augmented visual acuity of the F1 generation. Crossing the good eyesight strain and ha mutant produced fish having good eyesight and less sensitivity to gravity in the F2 population. Their tolerance to microgravity was tested by parabolic flight using an airplane. The fish exhibited less looping and no differences in degree of looping between light and dark conditions, suggesting that loss of eyesight (in darkness) is not a direct cause for looping behavior in microgravity. The ha embryos could not form utricular otoliths. They did form saccular otoliths, but with a delay. Fry of the mutant fish lacking the utricular otoliths are highly dependent on light upon hatching and exhibit a perfect dorsal-light response (DLR). As they grow, they eventually shift from being light-dependent to being gravity-dependent. Continuous treatment of the fry with altered light direction suppressed this shift to gravity dependence. Being less dependent on gravity, these fish can serve as models in studying the differences expected for the vestibular system of fish reared in microgravity. When these fish were exposed to microgravity (parabolic flights) of an airplane, they spent far less time looping than fish reared in an ordinary light regimen.     

Retinal photoreceptor and related gene expression in normal and clinostat-treated fish embryos

Medaka fish had performed mating behavior successfully in space for the first time among vertebrate, and the eggs which were laid in space developed normally, and hatched during the space travel of 15 days aboard the space shuttle in the second International Microgravity Laboratory (IML-2) mission in 1994 (Ijiri 1994). But there has been few studies whether microgravity affects the development of rather complex tissues in this fish. Investigating this problem, we focused on the organogenetic events in the retina in developing Medaka under normal and simulated microgravity conditions (by a three-dimensional clinostat, 3D-clinostat). Our results showed that both normal and 3D-clinostat-treated Medaka embryos developed on almost equal time course. Moreover, we investigated the development of the retina in normal and 3D-clinostat-treated embryos, but there were no differences in organogenesis of their retina. Lamination of retina occurred almost at equal timing and the expressions of opsin genes in the 3D-clinostat-treated group also began almost at the same time as control. Our observations suggest that there were no definite effects of simulated microgravity on the organizations of a complex tissue such as retina in developing fish embryos.     

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of Medaka fish.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.     

NASDA next generation aquatic habitat for Space Shuttle and ISS.

The National Space Development Agency of Japan (NASDA) has more than 20 years of experience developing aquatic animal experiment facilities. We are now studying the next-generation aquatic animal experiment facility or the Aquatic Habitat (AQH) for both Space Shuttle and International Space Station use. A prototype breeding system was designed and tested. Medaka adult fish were able to mate and spawn in this closed circulatory breeding system, and the larvae grew to adult fish and spawned on the 45th day after hatching. The water quality-control system using nitrifying bacteria worked well throughout the medaka breeding test. For amphibians, we also conducted the African clawed toad (Xenopus laevis) breeding test with the same specimen chambers, although a part of circulation loop was opened to air. Xenopus larvae grew and completed metamorphosis successfully in the small specimen chamber. The first metamorphic climax started on the 30th day and was completed on the 38th day.     

Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions

The influence of cosmic radiation and/or microgravity on insect development was studied during the 7 day German Spacelab Mission D1. Eggs of Carausius morosus of five stages differing in sensitivity to radiation and in capacity to regeneration were allowed to continue their development in the BIORACK 22°C incubator, either at microgravity conditions or on the 1 g reference centrifuge. Using the Biostack concept - eggs in monolayers were sandwiched between visual track detectors - and the 1 g reference centrifuge, we were able to separate radiation effects from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, growth kinetics and anomaly frequencies were determined in the different test samples. The early stages of development turned out to be highly sensitive to single hits of cosmic ray particles as well as to the temporary exposure to microgravity during their development. In some cases, the combined action of radiation and microgravity even amplified the effects exerted by the single parameters of space. Hits by single HZE particles caused early effects, such as body anomalies, as well as late effects, such as retarded growth after hatching. Microgravity exposure lead to a reduced hatching rate. A synergistic action of HZE particle hits and microgravity was established in the unexpectedly high frequency of anomal larvae. However, it cannot be excluded, that cosmic background radiation or low LET HZE particles are also causally involved in damage observed in the microgravity samples.     

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Otolith formation in a mutant Medaka with a deficiency in gravity sensing.

Mutant Medaka ha exhibit spontaneous mutation that is characterized by frequent inhibition or perturbation in the formation of utricular otoliths and/or semicircular canals. Three major features of otolith morphogenesis were observed in ha strain: 1) The initial appearance of otoliths was delayed, mispositioned, and malformed compared to normal embryos. 2) No utricular otoliths appeared on macula of any ha fry just after hatching. A symmetric state of otoliths was seen only when saccular otoliths were situated on macula in both inner ears. 3) In some fry, formation of utricular otoliths was observed in their later development. However, no new utricular otoliths appeared after fish were seventy or more days old after hatching. These observations show that otolith morphogenesis in ha is very different from that of wild-type. In this study, we classified adult ha into four different phenotypes using the existence or absence of utricular otoliths as our criteria. We concluded that dysfunction of utricular otoliths and semicircular canals cause a defect that affects the gravity-sensing abilities of medaka ha.     

Embryogenesis, hatching and larval development of Artemia during orbital spaceflight.

Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degrees of development can take place in this microgravity environment.     

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.     

Readaptation of fish to 1g after long-term microgravity: behavioural results from the STS 89 mission.

The swimming behaviour of adult and neonate swordtail fish Xiphophorus helleri was qualitatively analysed from video recordings taken throughout the STS 89 spaceshuttle mission from launch to landing and thereafter. After the flight, the swimming behaviour of neonate samples was quantitatively assessed in the course of the readaptation to 1g earth gravity at days 0, 1 and 4 after recovery. Regarding the swimming behaviour during the mission, the adult fish swam thigmotactically (i.e., responding to tactile stimuli) along the walls of their aquarium, but like the neonates, they did not show any aberrant behavioural patterns. This indicates that they could easily adapt themselves to microgravity. On mission day 9, however, looping responses (most probably initiated by mechanical disturbances) occurred indicating a continuously performed "C-start" escape response (the respective body bend looks like the letter "C"). Immediately after landing (observed in videos recorded onboard the space shuttle), the adults performed a head-up swimming beating heavily with the caudal and pectoral fins; this aberrant behaviour gradually decreased during the first hours after recovery.     

Effects of altered gravity on the swimming behaviour of fish.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness-phenomena (SMS, a kinetosis). It has been argued that SMS during PAFs might not be based on microgravity alone but rather on changing accelerations from 0 g to 2 g. We test here the hypothesis that PAF-induced kinetosis is based on asymmetric statoliths (i.e., differently weighed statoliths on the right and the left side of the head), with asymmetric inputs to the brain being disclosed at microgravity. Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated (1) whether or not kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry and (2) whether or not slow translational and continuously changing linear (vertical) acceleration on ground induced kinetosis. These latter accelerations were applied using a specially developed parabel-animal-container (PAC) to stimulate the cupular organs. The results suggest that the fish tested on ground can counter changing accelerations successfully without revealing kinetotic swimming patterns. Kinetosis could only be induced by PAFs. This finding suggests that it is indeed microgravity rather than changing accelerations, which induces kinetosis. Moreover, we demonstrate that fish swimming kinetotically during PAFs correlates with a higher otolith asymmetry in comparison to normally behaving animals in PAFs.     

Size and cell number of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness (SMS, a kinetosis). Since it has been repeatedly shown earlier that some fish of a given batch also reveal a kinetotic behavior during PAFs (especially so-called spinning movements and looping responses) and due to the homology of the vestibular apparatus among all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore, we examined the utricular maculae (they are responsible for the internalization of gravity in teleosteans) of fish swimming kinetotically at microgravity in comparison with animals from the same batch who swam normally. On the histological level, it was found that the total number of both sensory and supporting cells of the utricular maculae did not differ between kinetotic animals as compared to normally swimming fish. Cell density (sensory and supporting cells/100 micrometers2), however, was reduced in kinetotic animals (p<0.0001), which seemed to be due to malformed epithelial cells (increase in cell size) of the kinetotic specimens. Susceptibility to kinetoses may therefore originate in malformed sensory epithelia.     

A drop-tower experiment to determine the threshold of gravity for inducing motion sickness in fish.

It has been repeatedly shown earlier that some fish of a given batch reveal motion sickness (a kinetosis) at the transition from 1 g to microgravity. In the course of parabolic aircraft flight experiments, it has been demonstrated that kinetosis susceptibility is correlated with asymmetric inner ear otoliths (i.e., differently weighed statoliths on the right and the left side of the head) or with genetically predispositioned malformed cells within the sensory epithelia of the inner ear. Hitherto, the threshold of gravity perception for inducing kinetotic behavior as well as the relative importance of asymmetric otoliths versus malformed epithelia for kinetosis susceptibility has yet not been determined. The following experiment using the ZARM drop-tower facility in Bremen, Germany, is proposed to be carried out in order to answer the aforementioned questions. Larval cichlid fish (Oreochromis mossambicus) will be kept in a camcorder-equipped centrifuge during the microgravity phases of the drops and thus receive various gravity environments ranging from 0.1 to 0.9 g. Videographed controls will be housed outside of the centrifuge receiving 0 g. Based on the video-recordings, animals will be grouped into kinetotically and normally swimming samples. Subsequently, otoliths will be dissected and their size and asymmetry will be measured. Further investigations will focus on the numerical quantification of inner ear supporting and sensory cells as well as on the quantification of inner ear carbonic anhydrase reactivity. A correlation between: (1) the results to be obtained concerning the g-loads inducing kinetosis and (2) the corresponding otolith asymmetry/morphology of sensory epithelia/carbonic anhydrase reactivity will further contribute to the understanding of the origin of kinetosis susceptibility. Besides an outline of the proposed principal experiments, the present study reports on a first series of drop-tower tests, which were undertaken to elucidate the feasibility of the proposal (especially concerning the question, if some 4.7 s of microgravity are sufficient to induce kinetotic behavior in larval fish).     

Microgravity effects on Drosophila melanogaster development and aging: comparative analysis of the results of the Fly experiment in the Biokosmos 9 biosatellite flight.

The results are presented of the exposure of Drosophila melanogaster to microgravity conditions during a 15-day biosatellite flight, Biokosmos 9, in a joint ESA-URSS project. The experimental containers were loaded before launch with a set of Drosophila melanogaster Oregon R larvae so that imagoes were due to emerge half-way through the flight. A large number of normally developed larvae were recovered from the space-flown containers. These larvae were able to develop into normal adults confirming earlier results that Drosophila melanogaster of a wild-type constitution can develop normally in the absence of gravity. However, microgravity exposure clearly enhances the number of growing embryos laid by the flies and possibly slows down the developmental pace of the microgravity-exposed animals. Due to some problems in the experimental set-up, this slowing down needs to be verified in future experiments. No live adult that had been exposed to microgravity was recovered from the experiment, so that no life span studies could be carried out, but adult males emerged from the recovered embyros showed a slight shortening in life span and a lower performance in other experimental tests of aging. This agrees with the results of previous experiments performed by our groups.     

Pigment composition and concentrations within the plant (Ceratophyllum demersum L.) component of the STS-89 C.E.B.A.S. Mini-Module spaceflight experiment.

The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) Mini-Module, a Space Shuttle middeck locker payload which supports a variety of aquatic inhabitants (fish, snails, plants and bacteria) in an enclosed 8.6 L chamber, was tested for its biological stability in microgravity. The aquatic plant, Ceratophyllum demersum L., was critical for the vitality and functioning of this artificial mini-ecosystem. Its photosynthetic pigment concentrations were of interest due to their light harvesting and protective functions. "Post-flight" chlorophyll and carotenoid concentrations within Ceratophyllum apical segments were directly related to the quantities of light received in the experiments, with microgravity exposure (STS-89) failing to account for any significant deviation from ground control studies.     

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.     

Crickets in space: morphological, physiological and behavioral alterations induced by space flight and hypergravity.

"Crickets in Space" was a Neurolab experiment by which the balance between genetic programs and the gravitational environment for the development of a gravity sensitive neuronal system was studied. The model character of crickets was justified by their external gravity receptors, identified position-sensitive interneurons (PSI) and gravity-related compensatory head response, and by the specific relation of this behavior to neuronal arousal systems activated by locomotion. These advantages allowed to study the impact of modified gravity on cellular processes in a complex organism. Eggs, 1st, 4th and 6th stage larvae of Acheta domesticus were used. Post-flight experiments revealed a low susceptibility of the behavior to micro- and hypergravity while the physiology of the PSI was significantly affected. Immunocytological investigations revealed a stage-dependent sensitivity of thoracic GABAergic motoneurons to 3 g-conditions concerning their soma sizes but not their topographical arrangement. The morphology of neuromuscular junctions was not affected by 3 g-hypergravity. Peptidergic neurons from cerebral sensorimotor centers revealed no significant modifications by microgravity (micro g). The contrary physiological and behavioral results indicate a facilitation of 1 g-readaptation originating from accessory gravity, proprioceptive and visual sense organs. Absence of anatomical modifications point to an effective time window of micro g or 3 g-expo-sure related to the period of neuronal proliferation. The analysis of basic mechanisms of how animals and man adapt to altered gravitational conditions will profit from a continuation of the project "Crickets in Space".     

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.     

The mechanics of air-breathing in anuran larvae: implications to the development of amphibians in microgravity.

Because of their rapid development, amphibians have been important model organisms in studies of how microgravity affects vertebrate growth and differentiation. Both urodele (salamanders) and anuran (frogs and toads) embryos have been raised in orbital flight, the latter several times. The most commonly reported and striking effects of microgravity on tadpoles are not in the vestibular system, as one might suppose, but in their lungs and tails. Pathological changes in these organs disrupt behavior and retard larval growth. What causes malformed (typically lordotic) tadpoles in microgravity is not known, nor have axial pathologies been reported in every flight experiment. Lung pathology, however, has been consistently observed and is understood to result from the failure of the animals to inflate their lungs in a timely and adequate fashion. We suggest that malformities in the axial skeleton of tadpoles raised in microgravity are secondary to problems in respiratory function. We have used high speed videography to investigate how tadpoles breathe air in the 1G environment. The video images reveal alternative species-specific mechanisms, that allow tadpoles to separate air from water in less that 150 ms. We observed nothing in the biomechanics of air-breathing in 1G that would preclude these same mechanisms from working in microgravity. Thus our kinematic results suggest that the failure of tadpoles to inflate their lungs properly in microgravity is due to the tadpoles' inability to locate the air-water interface and not a problem with the inhalation mechanism per se.