Japanese red-bellied newts in Space--AstroNewt experiment on Space Shuttle IML-2 and Space Flyer Unit.

Biological effects of gravity was examined in embryonic development of Japanese red bellied newt. Two space newt missions were conducted in 1994 and 1995. The Second International Microgravity Laboratory was flown in 1994 as one of the SpaceLab missions. Space Flyer Unit, a Japanese space platform, was delivered to the earth orbit by the third launch of the H-II rocket and retrieved by Space Shuttle in 1996. Female newts were induced to lay eggs in orbit at these two space missions. Eggs were successfully obtained on both missions, and exposed to space environment from its early developmental stages. Morphology of the embryos was found not deviated from those developed on ground, as long as in the images taken in orbit or the examined specimen retrieved to ground. On the other hand, pathological changes were discovered in several organs of the adult newts that returned alive from their space flight.

Emesis and space motion sickness in amphibians.

Amphibians possess the ability to vomit in response to a variety of stimuli that provoke emesis in mammals. Pharmacological studies have establish that the ejection of gastric contents and the basic mechanism for vomiting have been phylogenetically conserved among these tetrapods. As part of on-going comparative studies on emesis in vertebrates, we previously documented that some postmetamorphic anurans and salamander larvae experience motion-induced emesis when exposed to the provocative stimulus of parabolic aircraft flight. However, more recent experiments suggest that there are strict conditions for inducing emesis in amphibians exposed to parabolic flight and that amphibians are not as sensitive to this stimulus as mammals. Further studies on emesis in lower vertebrates may help us understand the processes that cause emesis in abnormal gravitational regimes.

Frog experiment onboard space station Mir.

Japanese tree frogs (Hyla japonica) showed unique postures and behavior during an 8-day flight to the Russian space station Mir. When floating in the air, the animals arched their back and extended their four limbs. This posture resembles that observed during jumping or parachuting of the animals on the ground. Frog sitting on a surface bent their neck backward sharply, did not fold their hind limbs completely, and pressed their abdomen against the substrate. They walked backwards in this posture. The typical posture resembles that adopted during the emetic behavior process on the ground, although the posture in space lasts much longer. The possible mechanism of induction of this unique posture in orbit is discussed. Frogs in this posture might be in an emetic state, possibly due to motion sickness. Response behavior to some stimuli was observed in orbit. Body color change in response to the background color appeared to be delayed or slowed down. Response behavior to other stimuli showed little change as long as the animal maintained contact with a substrate. Once it left the surface, the floating frog could not control its movements so as to provide coordinated motility for locomotion and orientation. Adaptation to microgravity was observed in the landing behavior after jumping. Readaptation of the frogs to the Earth environment took place within a few hours after return. Postflight histological and biochemical analysis of organs and tissues showed some changes after the 8-day spaceflight. Weakening and density loss in vertebrae was noted. The beta-adrenoreceptor activity of the gastrocnemius was natriuretic decreased. Skin collagen and liver protein synthesis were lowered. The distribution of the atrial factor-like peptides in the brain was changed.

The Frog in Space (FRIS) experiment onboard Space Station Mir: final report and follow-on studies.

The "Frog in Space" (FRIS) experiment marked a major step for Japanese space life science, on the occasion of the first space flight of a Japanese cosmonaut. At the core of FRIS were six Japanese tree frogs, Hyla japonica, flown on Space Station Mir for 8 days in 1990. The behavior of these frogs was observed and recorded under microgravity. The frogs took up a "parachuting" posture when drifting in a free volume on Mir. When perched on surfaces, they typically sat with their heads bent backward. Such a peculiar posture, after long exposure to microgravity, is discussed in light of motion sickness in amphibians. Histological examinations and other studies were made on the specimens upon recovery. Some organs, such as the liver and the vertebra, showed changes as a result of space flight; others were unaffected. Studies that followed FRIS have been conducted to prepare for a second FRIS on the International Space Station. Interspecific diversity in the behavioral reactions of anurans to changes in acceleration is the major focus of these investigations. The ultimate goal of this research is to better understand how organisms have adapted to gravity through their evolution on earth.

AstroNewt: early development of newt in space.

AstroNewt experiment explores the effects of earth gravity on the early development of Japanese red-bellied newt, Cynops pyrrhogaster. Since female newts keep spermatophore in cloaca, fertilized eggs could be obtained without mating. Fertilization of newt's egg occurs just prior to spawning, so that gonadotrophic cues applied to females in orbit leads to laying eggs fertilized just in space. A property of newt being kept in hibernation at low temperature may be of great help for the space experiment carried out with much limited resources. A general outline of the AstroNewt project is shown here in addition to some technical advances for the development of the project. Experimental schemes of two space experiments (IML-2 in summer 1994 and unmanned SFU at the beginning of 1995) are also shown.

Cytopathologic observations of the lung of adult newts (Cynops pyrrhogaster) on-board the space shuttle, Columbia, during the Second International Microgravity Laboratory experiments.

Four adult female Japanese newts, Cynops pyrrhogaster, were carried for 15 days aboard the orbiting space shuttle, Columbia, in July of 1994, as part of the Second International Microgravity Laboratory, IML-2 aquatic animal experiments. These previously fertilized newts, after stimulation with chorionic gonadotropin by a spaceflight adapted injection procedure, deposited numerous eggs for study of early development during weightlessness. The primitive saccular lungs of the two newts which survived the spaceflight revealed by TEM marked pulmonary cytopathologic changes including basal laminar separation, microvillar degeneration, and cytoplasmic granular changes in the primary granulated pneumocytes. Also, intracellular edema in the pulmonary collagenous matrix and vacuolar changes in the ciliated pulmonary lining cell type and in vascular endothelial cells were observed. These changes, triggered by the spaceflight, and not seen in controls also relying on respiration via the skin, may reflect a chronic mild hypoxia as it is known that newts undergoing oviposition are subject to increased oxygen demand.

Amphibian amplexus in microgravity.

We report here on the amplectic behavior of the Japanese treefrog (Hyla japonica) in microgravity. Treefrogs were exposed to 35 cycles of altered gravity, including approximately 1.5 sec of G < 0.1 every 3 min and 15 sec, on the FreeFall "G.0" ride at Space World amusement park in Kitakyushu, Japan. During this period a pair of frogs spontaneously entered and maintained amplexus for 1 hr 20 min, before being removed from the ride. In freefall, the pair extended their hindlimbs in the characteristic posture of treefrogs in microgravity. This is the first report of a vertebrate entering and sustaining a copulatory or amplectic posture under gravitational extremes, including true freefall. These observations bode well for the potential of anurans to breed in microgravity and to be used for biological research in space.

Sustained release of hCG minipellet for newt experiment in space.

Sustained release formulation of hCG, hCG minipellet, was applied to induce oviposition of newt. Period of egg spawning was prolonged with a certain delay of its initiation. When hCG minipellet was injected to newt that was hibernating, it induced egg spawning even after one month of hibernation. Results suggest that minipellet keeps steady concentration of hCG at the effective level for longer period. For the study on early development of newt egg, it is essential to obtain egg on orbit. hCG minipellet makes it possible even at launch slip or early "late access".

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frog's adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.

Motion sickness in amphibians.

We explored the question of whether amphibians get motion sickness by exposing anurans (frogs) and urodeles (salamanders) to the provocative stimulus of parabolic aircraft flight. Animals were fed before flight, and the presence of vomitus in their containers after flight was used to indicate motion-induced emesis. None of the species that we studied vomited during the 8 to 10 parabolas of each flight. However, at least one specimen from each of the anuran species Rana rugosa, Rana nigromaculata, Hyla japonica, and Rhacophorus schlegelii vomited in a period of 0.5 to 42 h after flight. Some specimens of R. nigromaculata, H. japonica, and R. schlegelii were also observed retching without emesis either during or shortly after exposure to parabolic flight. We were unable to induce either emesis or retching behavior in the aquatic from Xenopus laevis. Among the urodeles studied we saw no signs of motion sickness in either adult or larval Cynops pyrrhogaster, but at least one larval Hynobius nebulosus vomited shortly after parabolic flight. The amphibian species that exhibited the most motion sickness were the same ones that showed the greatest amount of tumbling during the microgravity phases of their parabolic flights. The most distinctive difference between motion sickness in amphibians and mammals that vomit, including man, is the long delay between a provocative stimulus and emesis proper in the amphibians. The retching behavior we induced in the frogs was identical to that described previously for frogs treated with emetic drugs. H. japonica, exposed to extended periods of microgravity on the MIR Space Station, flattened their bellies against the substrate and dorsiflexed their heads in a manner reminiscent of drug-induced nausea. In light of our current observations of retching behavior in motion sick H. japonica, we suggest that the previously observed behavior of three frogs on the MIR Space Station was a manifestation of motion sickness.

The behavioral reactions of a snake and a turtle to abrupt decreases in gravity.

We report here on the behavioral reaction of two reptiles to abrupt decreases in gravity. One striped rat snake, Elaphe quadrivirgata, and three striped-neck pond turtles, Mauremys japonica, were exposed to microgravity on parabolic flight, during the filming of a documentary for the NHK television station in Japan. The video films revealed that the snake reflexively responded to the shift from hyper- to hypogravity by taking up a defensive posture--on the first parabola, the snake struck at itself. The turtles actively extended their limbs and hyper-extended their neck in microgravity, a posture which is identical to the displayed during their contact "righting reflex", when placed upside-down in normal gravity. The aggressive display of the snake was unexpected, although the righting response of the turtles was consistent with that shown by other vertebrates, including fish and mammals, exposed to microgravity. An implication of these observations is that the afferent signal for the righting reflex of vertebrates in normal gravity must be the unloading of ventral receptors in the sensory system, rather than the loading of dorsal receptors. These are the first behavioral records for any reptiles exposed to hypogravity.

Isolation and identification of intestinal bacteria from Japanese tree frog (Hlya japonica) with the special reference to anaerobic bacteria.

The bacteria in the large intestines of eight Japanese tree frogs (Hlya japonica) were enumerated by using an anaerobic culture system. The microorganisms at approximately 3.1 x 10(9) bacteria per g (wet weight) of intestinal contents were present in the intestine of all the frogs tested. No difference of the total bacteria in the frog intestine was observed between two different incubation-temperatures (room temperature and 37 degrees C). Eleven genera and 16 species were isolated from the frog intestine. In most frogs, Bacteroides (B.) caccae and B. vulgatus were detected as the predominant organisms. Escherichia coli was also present in greater numbers in the intestine. Other bacteria isolated at high dilutions were strict anaerobes, including Fusobacterium and Clostridium. Enterococcus faecalis was frequently isolated from the frog intestine. However, four genera of Bifidobacterium, Eubacterium, Peptostreptococcus, and Lactobacillus were not isolated from the frog intestine.

Space experiment on behaviors of treefrog.

Japanese treefrogs (Hyla japonica) are planned to be sent to the space station MIR. Experimental system was developed to observe their behaviors under microgravity.