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.     

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.     

Influence of hypergravity on fish inner ear otoliths: I. Developmental growth profile.

Inner ear stones (otoliths) of larval cichlid fish Oreochromis mossambicus were marked with the calcium-tracer alizarin-complexone (AC) at 1 g earth gravity before and after a 3, 7, 14 or 21 days stay of the animals at hypergravity conditions (hg; 3 g, centrifuge). After the experiment, the otoliths' area between the two AC-labellings was measured with regard to size and asymmetry (size difference between the left and the right stones). Both utricular and saccular otoliths (lapilli and sagittae, respectively) continued growing in a linear way at hg, but growth was significantly slowed down as compared to parallely raised 1 g-control specimens. In case of bilateral asymmetry between the corresponding otoliths its formation in hg-animals became reduced as compared to the 1 g controls. The reduction of asymmetry was much more pronounced in the sagittae than in the lapilli. The latter result supports an earlier hypothesis, according to which especially a low sagittal asymmetry has a functional advantage. In general, the results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector.     

Fish otolith growth in 1g and 3g depends on the gravity vector.

Size and asymmetry (size difference between the left and the right side) as well as calcium (Ca) content of inner ear otoliths of larval cichlid fish Oreochromis mossambicus were determined after a long-term stay at hypergravity conditions (3g; centrifuge). Both utricular and saccular otoliths (lapilli and sagittae, respectively) were significantly smaller after hyper-g exposure as compared to parallely raised 1g-control specimens and the absolute amount of otolith-Ca was diminished. The asymmetry of sagittae was significantly increased in the experimental animals, whereas the respective asymmetry concerning lapilli was markedly decreased. In the course of another experiment larvae were raised in aquarium hatch baskets, from which one was placed directly above aeration equipment which resulted in random water circulation shifting the fish around ("shifted" specimens). The lapillar asymmetry of the "stationary" specimens showed a highly significant increase during early development when larvae were forced to lay on their sides due to their prominent yolk-sacs. In later developmental stages, when they began to swim freely, a dramatic decrease in lapillar asymmetry was apparent. Taken together with own previous findings according to which otolith growth stops after vestibular nerve transaction, the results presented here suggest that the growth and the development of bilateral asymmetry of otoliths is guided by the environmental gravity vector, obviously involving a feedback loop between the brain and the inner ear.     

猫半规管破坏后线性加速度作用下耳石反应特点

本文研究线性加速度作用下,猫半规管阻塞前后前庭-眼动反射、前庭-脊髓反射和姿态平衡的反应特性。发现猫手术前和双侧半规管阻塞后耳石-眼动反应的功率谱和相对增益无显著性差异,脊髓反射H波幅变化在不同加速度刺激下有相似的反应模式。单侧半规管阻塞后,前庭-眼动反射表现明显不对称,H波幅改变与线性加速度无关,反映出半规管功能不对称也引起耳石功能不对称。本文提出潜在性耳石半规管功能不平衡可能是空间前庭功能障碍的重要原因。      

Calcium gradients in the fish inner ear sensory epithelium and otolithic membrane visualized by energy filtering transmission electron microscopy (EFTEM).

Inner ear otolith formation in fish is supposed to be performed by the molecular release of proteinacious precursor material from the sensory epithelia, followed by an undirected and diffuse precipitation of calcium carbonate (which is mainly responsible for the functionally important weight of otoliths). The pathway of calcium into the endolymph, however, still remains obscure. Therefore, the presence of calcium within the utricle of larval cichlid fish Oreochromis mossambicus was analyzed by means of energy filtering transmission electron microscopy (EFTEM). Electron spectroscopic imaging (ESI) and electron energy loss spectra (EELS) revealed discrete calcium precipitations, which were especially numerous in the proximal endolymph as compared to the distal endolymph. A decreasing proximo-distal gradient was also present within the proximal endolymph between the sensory epithelium and the otolith. Further calcium particles covered the peripheral proteinacious layer of the otolith. They were especially pronounced at the proximal surface of the otolith. Other calcium precipitates were found to be accumulated at the macular junctions. These results strongly suggest that the apical region of the macular epithelium is involved in the release of calcium and that calcium supply of the otoliths takes place in the proximal endolymph.     

Efficacy of an ototoxic aminoglycoside (gentamicin) on the differentiation of the inner ear of cichlid fish.

Previous investigations revealed that the growth of fish inner ear otoliths depends on the amplitude and the direction of gravity, thus suggesting the existence of a (negative) feedback mechanism. In the course of these experiments, it was shown that altered gravity both affected otolith size (and thus the provision of the proteinacious matrix) as well as the incorporation of calcium. It is hitherto unknown, as of whether sensory hair cells are involved either in the regulation of otolith growth or in the provision of otolithic material (such as protein or inorganic components) or even both. The ototoxic aminoglycoside gentamicin (GM) damages hair cells in many vertebrates (and is therefore used for the treatment of Meniere's disease in humans). The present study was thus designed to determine as of whether vestibular sensory cells are needed for otolith growth by applying GM in order to induce a (functionally relevant) loss of these cells. Developing cichlid fish Oreochromis mossambicus were therefore immersed in 120 mg/l GM for 10 or 21 days. At the beginning and at the end of the experimental periods, the fish were incubated in the calcium-tracer alizarin complexone (AC). After the experiment, otoliths were dissected and the area grown during GM-exposure (i.e., the area enclosed by the two AC labellings) was determined planimetrically. The results showed that incubating the animals in a GM-solution had no effect on otolith growth, but the development of otolith asymmetry was affected. Ultrastructural examinations of the sensory hair cells revealed that they had obviously not been affected by GM-treatment (no degenerative morphological features observed). Overall, the present results suggest that hair cells are not affected by GM concerning their possible role in (general) otolith growth, but that these cells indeed might have transitionally been impaired by GM resulting in a decreased capacity of regulating otolith symmetry.     

Allometry in vestibular responses of anurans

Frogs and toads turn either their heads or bodies opposite to angular accelerations applied around the yaw axis. Thresholds exist for the minimum angular acceleration that induces this vestibulomotor response in individual frogs. These thresholds were recorded for several anuran species that cover a broad range of sizes and life styles. Interspecific variation in the magnitude of the thresholds, which correlated with the ecology and behavior of the species, was documented. Also an allometric relationship was observed between this threshold and body size; the larger the frog, the lower the threshold. In many species, the threshold value for reflexive vestibulomotor responses to angular acceleration was proportional to the -0.4 (+/-0.2) power of body mass. Physical dimensions of the semicircular canals determine, in part, vestibular sensitivity to angular acceleration. Hence changes with growth in the semicircular canals are believed to contribute to the slope of -0.4. The biological significance of this allometry in vestibular responses is discussed and compared to trends in vestibular sensitivity and semicircular canal morphology of other vertebrate classes.     

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.     

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.     

Changes of vertical eye movements of goldfish for different otolith stimulation by linear acceleration.

Eye movements serves to hold the gaze steady or to shift the gaze to an object of interest. On Earth, signals from otoliths can be interpreted either as linear motion or as tilt with respect to gravity. In microgravity, static tilt will no longer give rise to changes in otolith activity. However, linear acceleration as well as angular acceleration stimulate the otolith organ. Therefore, during adaptation to microgravity, otolith-mediated response such as eye movements alter. In this study, we analyzed the eye movements of goldfish during linear acceleration. The eye movements during rectangular linear acceleration along the different body axis were video-recorded. The vertical eye rotations were analyzed frame by frame. In normal fish, leftward lateral acceleration induced downward eye rotation in the left eye and upward eye rotation in the right eye. Acceleration from caudal to rostral evoked downward eye rotation in both eyes. When the direction of acceleration was shifted 15 degrees left, the responses in the left eye disappeared. These results suggested that otolith organs in each side were stimulated differently.     

Influence of hypergravity on fish inner ear otoliths: II. Incorporation of calcium and kinetotic behaviour.

Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (hg; 3 g, 14 days) during development. Following the transfer to 1 g (i.e., stopping the centrifuge) they were separated into normally and kinetotically swimming individuals (the latter performed spinning movements). During hg, the animals were maintained in aquarium water containing alizarin-complexone (AC), a fluorescent calcium tracer. Densitometric measurements of AC uptake into inner ear otoliths (optical density of AC/micrometers2) revealed that the kinetotic individuals had incorporated significantly more AC/calcium than the normally behaving fish. Since the amount of otolithic calcium can be taken as an approximation for otolith weight, the present results indicate that the otoliths of kinetotically swimming samples were heavier than those of the normally behaving larvae, thus exhibiting a higher absolute weight asymmetry of the otoliths between the right vs. the left side of the body. This supports an earlier concept according to which otolith (or statolith) asymmetry is the cause for kinetoses such as human static space sickness.     

Swimming behavior of larval Medaka fish under microgravity.

Fish exhibit looping and rolling behaviors when subjected to short periods of microgravity during parabolic flight. Strain-differences in the behavioral response of adult Medaka fish (Oryzias latipes) were reported previously, however, there have been few studies of larval fish behavior under microgravity. In the present study, we investigated whether microgravity affects the swimming behavior of larvae at various ages (0 to 20 days after hatching), using different strains: HNI-II, HO5, ha strain, and variety of different strains (variety). The preliminary experiments were done in the ground laboratory: the development of eyesight was examined using optokinetic response for the different strains. The visual acuity of larvae improved drastically during 20 days after hatching. Strain differences of response were noted for the development of their visual acuity. In microgravity, the results were significantly different from those of adult Medaka. The larval fish appeared to maintain their orientation, except that a few of them exhibited looping and rolling behavior. Further, most larvae swam normally with their backs turning toward the light source (dorsal light response, DLR), and the rest of them stayed with their abdomen touching the surface of the container (ventral substrate response, VSR). For larval stages, strain-differences and age-differences in behavior were observed, but less pronounced than with adult fish under microgravity. Our observations suggest that adaptability of larval fish to the gravitational change and the mechanism of their postural control in microgravity are more variable than in adult fish.     

Mass and mechanical sensitivity of otoliths.

It has been suggested that the changes of otolith mass during the otolith development in altered gravity conditions as well as the growth of otoliths in fishes in normal conditions are determined by the feedback between the otolith dynamics and the processes that regulate otolith growth. This hypothesis originates from the pendulum model of an otolith (de Vries, 1950), in which otolith mass is a parameters. The validity of this hypothesis is tested by comparing the pendulum model with a simplified spatially distributed model of an otolith. It was shown that when the otolith plate (otoconial layer) was spatially distributed and fixed to the macular surface, the mechanical sensitivity of the otolith does not depend on the total otolith mass and its longitudinal dimensions. It is determined by otolith thickness, Young's modulus, and the viscosity of the gel layer of the growing otolith. These parameters may change in order to secure otolith sensitivity under altered dynamic conditions (e.g., in microgravity). Possible hypotheses regarding the relationship between the otolith growth, otolith dynamics and animal growth are proposed and discussed here.     

Effects of simulated weightlessness on fish otolith growth: Clinostat versus Rotating-Wall Vessel

Stimulus dependence is a general feature of developing sensory systems. It has been shown earlier that the growth of inner ear heavy stones (otoliths) of late-stage Cichlid fish (Oreochromis mossambicus) and Zebrafish (Danio rerio) is slowed down by hypergravity, whereas microgravity during space flight yields an opposite effect, i.e. larger than 1 g otoliths, in Swordtail (Xiphophorus helleri) and in Cichlid fish late-stage embryos. These and related studies proposed that otolith growth is actively adjusted via a feedback mechanism to produce a test mass of the appropriate physical capacity. Using ground-based techniques to apply simulated weightlessness, long-term clinorotation (CR; exposure on a fast-rotating Clinostat with one axis of rotation) led to larger than 1 g otoliths in late-stage Cichlid fish. Larger than normal otoliths were also found in early-staged Zebrafish embryos after short-term Wall Vessel Rotation (WVR; also regarded as a method to simulate weightlessness). These results are basically in line with the results obtained on Swordtails from space flight.     

No correlation between multilamellar bodies in the inner ear and further organs of mutant (backstroke, bks) and wildtype zebrafish embryos.

The origin of the proteinacious matrix of the inner ear stones (otoliths) of vertebrates has not yet been clarified. Using the backstroke mutant (bks) of the zebrafish Danio rerio, which is characterized by a complete lack of otoliths, we searched for possibly missing or aberrant structural components within the macular epithelia of the inner ears of embryos on the ultrastructural level. Numerous multilamellar bodies (MLBs) were found. The MLBs were, however, not restricted to the inner ears of mutants but were also found in wildtype individuals and in further organs such as brain and liver. MLBs have hitherto never been described from the inner ear of fish and are generally estimated to be rare structures. Their occurrence in fish liver can, however, be induced by using particular chemical substances, which seem to effect adaptive compensatory processes on the cellular level. Such a chemical treatment also affects the ultrastructure of further organelles. Since the occurrence of MLBs in the liver of zebrafish was not accompanied by an alteration of the morphology of other organelles, their occurrence seems not to be due to environmental stress. The findings indicate that the MLBs cannot be correlated with bks-inherent features as well as with missing otolith development/growth. Since the occurrence of MLBs was independent from the developmental stage of a specimen and its overall tissue preservation, it can moreover be excluded that these MLBs merely represent fixation artifacts. Their presence more likely indicates cellular remodelling processes of hitherto unknown significance.     

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).     

Calcium-tracers disclose the site of biomineralization in inner ear otoliths of fish.

Since changing gravity (concerning direction and amplitude) strongly affects inner ear otolith growth and otolithic calcium incorporation in developing fish, it was the aim of the present study to locate the site of mineralization in order to gain cues and insights into the provenance of the otoliths inorganic compounds. Therefore, larval cichlid fish (Oreochromis mossambicus) were incubated in the calcium-tracer alizarin complexone (AC; red fluorescence). After maintenance in aquarium water for various periods (1, 2, 3, 6, 9 and 12 h; 1, 2, 3, 5, 6, 7, 15, 29, 36 and 87 d), the animals were incubated in the calcium-tracer calcein (CAL; green fluorescence). AC thus labeled calcium being incorporated at the beginning of the experiment and would subsequently accompany calcium in the course of a possible dislocation, whereas CAL visualized calcium being deposited right at the end of the test. Subsequently, the otoliths were analyzed using a laser scanning microscope and it was shown that the initial site of calcium incorporation was located directly adjacent to the sensory epithelium and the otolithic membrane. Later, calcium deposits were also found on further regions of the otoliths' surface area, where they had been shifted to in the course of dislocation. This finding strongly indicates that the sensory epithelium plays a prominent role in otolithic biomineralization, which is in full agreement with an own electron microscopical study [ELGRA News 23 (2003) 63].     

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.     

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.