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Chapter 8 Frog Experiment Onboard Space Station Mir
€hapter 8
FROG EXPERIMENT ONBOARD SPACE STATION MIR Akem i IzumLKurotani. Yosh ihiro Mogami. Makoto Okuno. and Masamichi Yamashita I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Experimental System and Operation . . . . . . . . . . . . . . . . . . . . . . A . TreeFrog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Transportation of Frogs to Mir . . . . . . . . . . . . . . . . . . . . . . . C . Experiment in Orbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Recovery of Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Ground Control Experiment . . . . . . . . . . . . . . . . . . . . . . . . 111. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Experimentinorbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Control Experierntn on Ground . . . . . . . . . . . . . . . . . . . . . . C. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Space Biology and Medicine Volume 6. pages 193-211 Copyright 0 1997 by JAI Press Inc All rights of reproduction in any form reserved ISBN: 0-7623-0147-3
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1. INTRODUCTION Living creatures on earth have evolved in the earthly environment from birth. Gravity is one of the important parameters in the environment. Behavior of various animals on this planet is influenced by gravity. Some space experiments have been aimed at the study of the role of gravity in animal behavior, for instance in the construction of beeswax combs in a colony of honey bees,' in the mating behavior of parasitic wasps,' in the web-building behavior of garden cross spiders? in the swimmingbehavior ofjelly fish' and killifi~h,'.~ in the swimming behavior of larva and adult of African clawed frog? and in the behavior of space-hatched Japanese
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In December 1990, we sent six Japanese tree frogs to the Russian space station Mir to investigate their posture and behavior in microgravity. Tree frogs present several types ofbehavior, i.e., walking,climbing,swimmingandjumping. Principal objective of this space experiment was to study how the frogs would respond to the condition of weightlessness. When a frog is briefly exposed to microgravity in free fall (1-2 seconds) or parabolic flight (about 20 seconds)? they arch their back and stretch out their four limbs. Spaceflight can greatly extend the duration of exposure to microgravity, in this mission to 8 days. In this experiment we observed the posture and behavior of Japanese tree frogs during a prolonged stay in the microgravity environment. Questions studied were, for example: When frogs try to change their position, do they select any original mode of behavior in microgravity?; Can they create a new way of locomotion? When an external stimulus is presented to the animals in orbit, is the stimulus strength changed from that observed on earth?
II. EXPERIMENTAL SYSTEM AND OPERATION A. Tree Frog
The Japanese tree frog, Hylujuponicu,is an arboreal animal, i.e., it normally lives on leaves and twigs of small grasses or shrubs. During the breeding season it appears in or near the water, such as in a wet rice field. Its fingers have round adhesive discs and poorly developed webs.' From a group of about 400 tree frogs, caught in fields in the Kanto area of Japan, 100 healthy specimens were selected for the experiment. One of the criteria for selection was the ability of the animals to change their body color relatively rapidly (within 10 minutes; see section III).' Of these animals 18 were transported to the training site Star City, Russia, in advance and kept there. The remaining 82 animals were transported to the launch site, 10 days before launch, and were added to the advance group. The ability to change body color rapidly was rechecked at the launch site. The age of the animals ranged from 7-1 2 months, and their body weight from 3-1 u
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Table 1. Selection Criteria for Animals 1 . Absence of visible injuries
2. Health condition expressed by posture Frogs in poor health often crouch. 3. Normality of vestibular functions Optokinetic nystagmus: Head rotation was observed when the stage on which a frog sits turns on dorm-ventral axis with angular velocity of 20-45 degreedsec. Gravity response: Reorientation of head was observed when stage on which frog sits was tilted to 3 0 4 5 degrees from horizontal. Two direction (right-left and anterior-posterior axis) were tested. Righting response: Righting response was observed after a frog was made to lie on its back by an operator’s hand.
The day before launch 12 frogs (six males and six females) were selected from the group of 100 animals by means of the criteria listed in Table 1. The 12 frogs were divided into two groups, six flight animals and six ground control animals. All animals were starved during the 10-day period preceding launch. This was done to prevent clogging of the air vents of the Life Support Box with feces. In orbit the frogs were not fed routinely, but they could eat meal worms, which were brought to serve as food in the experiment on feeding behavior. The intestinal microflora of the Japanese tree frog was analyzed in advance to ensure that the safety requirements for the cosmonauts would be met.’
B. Transportation of Frogs to Mir A Life Support Box, shown in Figure 1, was used for transportation of the living specimens to Mir. It travelled on the Soyuz, which stayed in transfer orbit for two days before docking to Mir. The Life Support Box has a three-layered structure. The top layer has seven compartments: six for one flog each, and one for 30 meal worms (living larvae of Tenebrio obscurus) serving as food inflight. The frog compartment snugly fits a sitting frog and is lined with polyurethane foam, so as to protect the animal against the vibrations and shocks during launch. The foam is water-soaked to keep the animal from dehydrating. The compartment for meal worms is not lined with polyurethane foam and is kept dry. During transportation the frogs cannot approach the meal worms. Zeolite in the top layer absorbs odor and urine from the living specimens. Each frog was installed in its compartment no less than 7 hours before launch. The middle and bottom layers of the Life Support Box house a small air pump and air ducts. The small air pump is powered by a Ni-Cd battery. The air ducts are made of soaked polyphenol foam to keep the air moist for frogs. There are two air
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Figure 1. Life Support Box used for transportation of frogs on Soyuz, which stayed in transfer orbit for two days before docking to Mir.
inlets in the bottom layer and two outlets in the top layer. Membrane filters with sub-micron pores are present in the air inlets and outlets for the purpose of bioisolation. Fresh cabin air was drawn into the Life Support Box by the air pump for 2 days during transportation to Mir. The temperature in the box was passively kept at the level of the crew cabin. C. Experiment in Orbit
The Frog Observation System was used for the experiment in orbit in Mir. It is composed of a glove bag for the animals (Figure 2), a tool bag with two sets of experiment tools (Figure 3), a CCD camera and an 8 mm videorecorder. It was shipped to Mir on the unmanned Progress, about 2 months prior to the anival of the frogs. Of the two sets of experiment tools, one set was for use inside the glove bag, the other for use outside the glove bag. A complete list of the two sets of tools is presented in Table 2. Tools were kept in pockets of the tool bag to prevent their scattering in weightlessness. On the day after rendezvous of S o p and Mir (three days after launch = day L + 3) the frogs were transferred from the Life Support Box to the glove bag. First, the Life Support Box with the living specimens, and the tool band with experiment tools to be used inside the glove bag, were placed inside the glove bag. They were
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Figure 2. Glove bag for observation and handling of frogs in bioisolation. fixed on the inner face of the glove bag by Velcro tape. Second, the glove bag was sealed by a seal clip bar, and inflated by air released from a small air cylinder. A polyurethane foam on the tool band was soaked with water from a water reservoir tube to keep the inside moist. Then the frogs were set free in the glove bag for observation, and were kept in this bag during the entire mission. Frogs and experiment tools in the glove bag were handled through gloves. The health condition of the frogs (alive, weakened, or dead) was checked daily by visual inspection for a few minutes. From that point observation of the animals was started. The experiment with observation and recording of the frogs was performed twice, on days L+3 and L+5, in order to determine whether any form of adaptation occurred. The subjects of observations are listed in Table 3. On the experiment days the behavior of the frogs was recorded with a small CCD camera or 8-mm VTR camera by a Japanese mission operator or a Russian cosmonaut.
D. Recovery of Frogs AFrog Recovery Box (Figure 4) was installed in the tool band, which was placed inside the glove bag at the beginning of the experiment. On the last day of the mission (day L+8) an appropriate volume of water was poured on a calcium peroxide tablet in the Frog Recovery Box to generate suficient oxygen gas for life support during the recovery phase. The polyphenol foam covering the interior
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Table 2. List of Tools a. Tools used outside Glove bag Name of Tool
Quantity
Seal clip bar Hand-powered Air Pump Air cylinder Pressure regulator and valve
3
Audio cassette tape Supporting band
1
Camera holder Scissors Mending tape Inner gloves
1 1 1 1
Gloves in exchange
1
1 1
1
1
Use for Sealing of glove bag Air withdrawal from glove bag at end of expt. Inflating glove bag at start of expt. Regulation of releasing pressure of air from air cylinder Voice stimuli (not used) Fixing position of observation window to the cosmonaut Fixing CCD camera head on observation window Cutting off part of glove bag for frog recovery Mending of damage of glove bag (not used) Absorption of sweat of hands in gloves of glove bag (not used) Gloves of glove bag for back-up cosmonaut (not used)
b. Tools used inside Glove bag Name of Tool
Quantity
Water reservoir tube Air blower Suction pipette Tweezers
4
Scissors Wrench-driver Mending tape Vinegar bottle Imitative snake Imitative meal worm Imitative willow sprig Needle (in Needle holder) Paper for wipe Paper for white balance Burial bag Burial box Polyurethane foam Joint tube
1 1 1 1
Frog recovery box
2 1 1
1 1 1
1 3 1 5 1
1 1
1
Use for Container of water Positioning of frog (not used) Positioning of frog (not used) Handling of vinegar-immersed filter paper (not used). Mechanical stimuli (not used) Picking up meal worm General use Screwing off of fuse of LSB Mending of damage of gloves (not used) Experiment of chemical stimuli (not used) Experiment of visual stimuli (not used) Experiment of visual stimuli Experiment of visual stimuli Euthanasia (not used) Wipe of observation window (not used) Adjusting white balance (not used) Keeping carcass Keeping burial bag with carcass Humidifier Connecting between LSB and outlet of glove bag to use Zeolite in LSB as deodorizer for gas from glove bag Recovery of frogs
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Figure 3. Tool bags with tools used outside (a) and inside (b) glove bag.
surface of the frog compartment in the Frog Recovery Box was soaked with water to provide moisture for the animal. The six frogs were placed in the individual frog compartments. The Frog Recovery Box was removed from the glove bag without breaking bioisolation by the technique shown in Figure 5 . The box was placed in the entrance port of the glove bag. The port was parted by two seal clip bars, which were placed
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Table 3. Subjects of Observations of Frogs 1. Behavior and posture in microgravity 2. Response behavior to various external visual stimuli real food (a living meal worm) small moving object (imitation willow sprig with leaves, imitation worm or tweezers) large moving object (hand of operator) 3. Orientation behavior on rotating object (water reservoir tube) 4. Ability to change body color
between the box and the body of glove bag. After cutting the space between the two seal clip bars by a scissors, the Frog Recovery Box was isolated from the glove bag and encapsulated. Each cut end was wiped with a sterilizing cloth. The encapsulated Frog Recovery Box was returned to the ground in the Soyuz recovery capsule. E. Postflight Experiments Behavior and Vestibular Function
Observation and recording of behavior after recovery were performed in a hall of the airport closest to the landing site, as early as two hours after landing. The process of jumping, landing, walking, and climbing a wall by the frogs was observed and recorded. The vestibular function was also tested. Test items and procedures were the same as those at the launch site before launch (see Table 1). These observations were repeated again at 12 hrs after landing.
Figure 4. Frog Recovery Box used for recovery of frogs upon return to Earth.
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figure 5. Procedure for isolation of Frog Recovery Box from the glove bag.
Preparation of Organs and Tissues From 2.5 hours after landing, two spaceflight frogs and two ground-control frogs were dissected and their organs and tissues were fixed by a procedure suitable for later analysis. The outline of the entire experimental procedure is diagrammatically represented in Figure 6 .
F. Ground Control Experiment Ground-control frogs were kept for about 2.5 days in an identical Life Support Box as used for the transport of the flight animals. They were then released in an air-opened box (200W x 125D x 140H mm) at the same time as the flight frogs were released in the glove bag on the Mir station. Temperatureand humidity in the
R
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Lafe Access
LSB
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LSB
@ 1st EXD.L+3 davs 2nd Ex'p. L+5 da'ys
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Recovery
FRB FRB
R+(-2.5 hrs)
Observation Disection
R+(-2 hn) Early Access
R+(-lh)
C CI l>l >
B
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-1
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Figure 6. Outline of operation of the Frog Experiment.
LSB : Life Supporl Box 2.5 days -4-
SOYUZ
?3b
e
LSB
LSB
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two containers did not exactly simulate those in the flight Life Support Box and the glove bag in the Mir station. The control frogs were then placed in a Frog Recovery Box identical to that used inflight in reserve, and kept there for about 1 day. Behavior and vestibular function were observed and recorded on days L+3 and L+5, and after return of the flight animals.
111. RESULTS AND DISCUSSION A. Experiment in Orbit
Recorded video tapes were handed to us ten days after return (day R+10). The Japanese mission operator was interviewed about one month after his return. From the tapes and the interview the following was learned. Behavior of Floating Frogs
Frogs floating in the air inflight showed a posture similar to that during the few seconds of microgravity in a parabolic flight or free They arched their back, inflated their abdomen, and extended the four limbs with opened fingers and toes (Figure 7). During floating, synchronous movements (like breaststroke swimming or scissors-kick) and asynchronousmovements of both hind limbs were observed sometimes. These movements lasted for such a short period that they could not provide a system of coordinated motility for locomotion. Floating frogs tried to catch whatever object one of their four limbs touched. The limbs pointed in a rather
Figure 7. Behavior of a frog floating in weightlessness. Arched back, inflated abdomen, extended limbs with fingers and toes opened and hind limbs extended in lateral position.
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lateral direction, although the direction was not stable. The typical posture during floating in microgravity resembled, except for the position of the limbs, the posture observed during jumping on the ground (Figure 8). It has been reported that at dawn arboreal sub-tropical forest frogs parachute from a canopy of rain forest to the ground.lo The parachuting frogs “move their front and hind limbs lateral to their bodies and spread their fingers and toes during aerial descent, adjusting limb position slightly during flight....... this lateral position of the limbs is quite different than that observed during the trajectoryphase of a regular jump where the forelimbs are anterior and lateral but the hind limbs remain extended posterior to the body.”” According to this description, the parachuting posture appears to bear a close resemblance to the floating posture in orbit. However, it is not known whether the Japanese tree frog makes a retarded descent like the subtropical forest frog. They may do it in a small way from shrubs or grasses. Further observation and analysis are required to clarify this point. This parachuting-like posture in orbit is not a common response to microgravity among frogs and toads. Recently we have been investigating the behavioral response of various frogs and toads to an abrupt decrease in gravity in free fall and parabolic These experiments suggest that the response can be related to
Figure 8. Jumping posture of frog postflight. Frog was made to jump from a platform (15 mm height) and land on polyurethane sheet. Recorded in darkness by 35-mm camera with multiple exposures in strobe light (0.1 sec. intervals). Frog extended its hind limbs backwards during the iump.
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their way of life.14In microgravity these frogs and toads, who are non-arboreal and move two-dimensionally on the ground surface (Rana rugosa, Rana nigromaculata, Xenopus laevis, Lepidobatrachus budgetti and Ceratophryssp.),tend to rotate around their rostral-caudal axis by means of scissor-like kicks. This long axis rotation is similar to their righting reflex when the animal is inverted in normal gravity, while arboreal or sub-arboreal frogs (Hyla japonica and Rachophorus schlegefii) often show a similar posture during parachuting. Behavior of Stationary Frogs
Though some frogsjumped off spontaneously,most of them did not move, stayed on a surface, or hid themselves under some object during the mission. Frogs frequently stayed on a moist polyurethane foam. Some frogs returned to a compartment in the Life Support Box in the glove bag. When a frog was sitting on a surface, like the glove bag, they frequently (four out of six frogs) assumed a particular posture. The neck was sharply bent backwards (at nearly right angle), the back was arched, the hind limbs were not folded completely, and the abdomen was pressed against the substrate (Figure 9). In this posture they would walk backwards. Posture in Microgravity
We have studied what this posture might express. The effects of selective neurectomy in the labyrinth on motor function has been investigated by several groups.15-'* This procedure might simulate the behavior experiment in microgravity, since the input of the gravity signal can be prevented by this neurectomy.
Figure 9. Typical posture of a frog sitting on surface of glove bag. Arched back, incompletely folded hind limbs, abdomen pressed against glove bag.
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Figure 10. Posture during emesis induced by CuSO4 solution. 10 pI CuSO4 solution (0.2 mg per g body weight) was administered by gastric cannula (photograph by Dr. T. Naitoh).
The posture and behavior observed after neurectomy do not directly explain the particular posture observed in Mir. Naitoh et aLt9 have reported that emetic behavior (vomiting and retching: emetic behavior in the absence of any regurgitation) can be induced in frogs by certain drugs. The particular posture in orbit resembles the posture during retching behavior induced by emetic drugs (Figure 10). We have subjected several species of frogs to parabolic flights in order to test whether frogs can suffer from motion sicknessby mechanical acceleration.12Some frogs showed signs of emetic behavior, like cyclical mouth opening and closing, blinking, and walking backwards during the flight, some frogs actually vomited after the flight. Thus we have concluded that frogs, including Hyla japonica, can suffer motion sickness from parabolic flight. Cyclical mouth opening and closing of the frogs was also observed by the mission operator in Mir.20The frogs showing the particular posture observed in Mir may thus have been in an emetic state, possibly due to motion sickness. Response Behavior
The body color of each frog was observed soon after their release from the Life Support Box into the glove bag. The color of the polyurethane foam lining of three compartments in the box was white, of the other three black. On the ground the frogs respond to the white color by a change of their body color to yellow-green within 10 min., to the black color by a change to green-black. According to the report of the mission operator,21one of the frogs from a white compartment turned dark soon after its release into the glove bag. This observation suggests that the
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body color change response to the background color may be inhibited in space. It was not clear whether the response was only delayed, since the mission operator made no later report about the body color. Another point is that the body color change depends on the light intensity, and it was not known whether the light intensity at the site of the experiment in Mir was sufficient to induce the body color change. The mission operator did not report about the colors of the frogs that had been transported in black compartments. When the mission operator brought his hand to the glove bag, he noticed an avoiding response as usually made from a large object. When the mission operator placed a meal worm in front of a frog sitting on the surface of the glove bag, the frog tried to catch it by a forefoot, but it failed to catch the worm. This failure seemed to be due to an instability of the footing when the frog put out its forefoot. When a small (95 mm length) pair of tweezers floated while rotating along its long axis, one frog looked toward it and oriented its body to it, however it could not approach the tweezers after jumping off. It seems that frogs in microgravity succeed in making a response behavior only when they keep a stable contact with a surface by means of the round adhesive discs on their fingers. Once they left the surface and floated, they could not control their movements sufficiently to respond to a stimulus. They could only approach a food object and eat it by keeping a steady contact with a surface. The hvo frogs, who were dissected after recovery, were found to have some food (meal worms?) in their stomach, although the mission operator could not observe eating behavior in orbit. Previously, it has been reported that space-hatchedJapanese quails could not approach their food without the help of a c~smonaut.~ Quail chicks might not be able to hold on to the substrate while moving toward their food. When a frog rode on a rotating water reservoir tube (220 mm length, 32-50 mm diameter), it walked along the circumference toward the opposite direction. The orientation behavior observed on the ground was also shown in orbit, however it is not clear whether the frogs oriented themselves by a visual standard, or by angular acceleration sensing, or both. Adaptation to Microgravity
After a frog jumped off in orbit, it would try to land or perch on some object, but often failed to do so. The frequency of failure to land or perch after ajump decreased with time: from an average of 2.7 random contacts with an object or surface per jump before proper landing on day L+3 to an average of 1.4 contacts per jump before landing on day L+5. This finding suggests that frogs can adapt to microgravity in the matter of landing after a jump. However, the typical posture while sitting on a surface and the parachuting-like posture during floating were maintained until the end of the experiment. Adaptation to microgravity in the behavior of other animals has been reported. A cross spider could build a better web after 3 weeks in microgravity than in the
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beginning of its stay in orbit.2 The frequency of looping behavior of killifish in microgravity gradually decreased after staying in orbit for several days.'33 B. Control Experiment on Ground
When the ground-control frogs were released from the Life Support Box or the Frog Recovery Box, their walking, climbing, swimming and jumping behavior and vestibular function (optokinetic nystagmus, gravity response and righting response) were normal. C. Postflight Experiments Behavior of Recovered Frogs
All six frogs were recovered and returned alive after eight days of spaceflight. Upon the first observation of the animals, two hours after return (R+2 hrs.), all six frogs walked slowly and climbed a wall toitteringly. After landing from a jump, the folding of the hind limbs was delayed. Vestibular function did not appear clearly. Half an hour later (R+2.5 hrs.), their behavior and vestibular function began to return to normal. At R+12 hrs. behavior and vestibular function were normal again. Abnormal swimming behavior (looping) in Xenopw larvae has been reported to continue for 1-2 days after their recovery from a 7-day pacef flight.^ We cannot conclude that readaptation to 1-G condition in adult tree frogs was faster, since there are few reports about the behavior of adult frogs and toads after recovery from spaceflight. Analysis of Organs and Tissues
The organs and tissues of recovered and ground-control specimens were distributed to several research groups for a histological and biochemical analysis of the effect of an 8-day spaceflight. Their findings are summarized below. Kashima et al. observed the weakening of a vertebral bone.22The spongy bone in the caudal articulatio vertebra was less dense in the spaceflight animals than in the ground-controls.In a quantitative analysis by means of a bio-imaging analyzer they found 20% less calcium density in the posterior joint of the seventh vertebra of a spaceflight frog. Ohira and coworkers found a decrease in the P-adrenoreceptor activity, which is thought to correspond to mitochondria1biogenesis, in the gastrocnemiusof spaceflight frogs.23 Yamazaki and coworkers found a decreased collagen content in the skin and a lowered protein synthesis in the liver of spaceflight Suzuki and coworkers studied the morphology of the vestibular sensory epithelium by means of scanning electronmicroscopyand light micro~copy.~~ No changes
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were found in the sensory cilia of the utricular macula and the semicircular canal cristae in spaceflight frogs. Feuilloley and coworkers examined in heart and brain of spaceflight frogs the distribution of atrial natriuretic factor (ANF)-like peptides, which regulate water and electrolyte balance and blood pressure.26327 The density and distribution of the staining were identical in the hearts of control and spaceflight frogs. In the ground-control frogs ANF-positive cell bodies were found in the parium and striatum of the telencephalon, the lateral forebrain bundle of the diencephalon, and the nucleus reticularis isthmi in the mesencephalon. These were absent in the spaceflight frogs. Conversely, ANF-immunoreactivity was observed in the posterior nuclei of the posterolateralis thalamus in spaceflight frogs, but in the groundcontrol frogs these nuclei were scarcely stained. The authors suggested that prolonged exposure to microgravity affects biosynthesis and/or release of ANFrelated peptides in discrete regions of the frog brain.
IV. CONCLUSIONS AND SUMMARY Japanese tree frogs (Hylajaponica) showed unique postures and behavior during an 8-day flight on 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. Frogs 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. This 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 external 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 P-adrenoreceptor activity of the gastrocnemius was decreased. Skin collagen and liver protein synthesis were lowered. The distribution of the atrial natriuretic factor-like peptides in the brain was changed.
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ACKNOWLEDGMENTS We thank the persons i n Japan and Russia, whose efforts made the “Frog I n Space” project possible. A partial list of names is provided in reference 29. T h e extended studies on the postflight behavior of frogs and toads would not have been possible without the cooperation of Dr. R.J. Wassersug and Dr. T. Naitoh.
REFERENCES 1. Stark, R.E. Ethology in Space, a Unique Opporfunity for Behavioral Science. ESA STM-246,
ESA, Paris, 1993. 2. von Borstel, R.C., Smith, R.H., Whiting, A.R., Grosch, D.S. Mutational and physiologic responses of Habrobracon in Biosatellite 11. In: The Experiments of Biosatellite 11 (J.F. Sanders, Ed.), pp. 17-39. NASA SP-204, NASA, Washington, D.C., 1971. 3. Scheld, H.W. Baky, A., Boyd,J.F.,Eichler,V.B.,Fuller,P.M.,Hofhan,R.B., Keef, J.R., Kuchnow, K.P., Oppenheimer, G.M., Salinas, G.A., von Baumgarten, R.J. Killifish hatching and orientation. In: Apollo-Soyw Test Project Surnmaiy Science Report (NASA Editorial Review Board), Vol. I , pp. 281-305. NASA SP-412, NASA, Washington, D.C., 1977. 4. Briegleb, W., Neubert, J., Schatz, A., Klein, T., Kruse, B. Survey of the vestibulium and behavior of Xenopus laevis larvae developed during a 7-day spaceflight. Advances in Space Research, 6(12):151-156, 1986. 5. The quails that flew in space In: USSR Space Life Sciences Digest, issue 30 (L.R. Stone, R. Teeter, J. Rowe, Eds.), pp. 37-40. NASA CR-3922(36) NASA, Washington, D.C., 1991. 6. Izumi-Kurotani, A., Yamashita, M., Kawasaki, Y., Mogami, Y., Okuno, M., Oketa, A. Behavior of tree frogs under microgravify. Biological Sciences in Space, 5(3):185-1 89, 1990. 7. Maeda, N., Matsui, M. Frogs and Toads of Japan, Bun-Ichi Sogo Shuppan, Co., Tokyo, 1989 (Japanese). 8. Akimoto, M., Kawamura, K., Namiki, H., Kikuyama, S. Evaluation ofadaptability of“space frog” candidates to background color. Biological Sciences in Space, 5(3):212-2 14, 1991. 9. Benno, Y., Izumi-Kurotani, A,, Yamashita, M. Intestinal microflora ofjapanese tree frog (Hyla japonica). Biological Sciences in Space, S(3):182-1 84, 1991. 10. Stewart, M.M. Arboreal habitat use and parachuting by a subtropical forest frog. Journal of Herpetology, 19(3):391-40 1, 1985. 11. Emerson, S.B., Koehl, M.A.R. The interaction of behavioral and morphological change in the evolution of a novel locomotor type:“flying” frogs. Evolution, 44(8):193 1-1946. 1990. 12. Wassersug, R.J., Izumi-Kurotani, A., Yamashita, M., Naitoh, T.Motion sickness in amphibians. Behavioral and Neural Biology, 60:42-5 1, 1993. 13. Izumi-Kurotani, A., Wassersug, R.J., Yamashita, M., Naitoh, T., Nagaoka, S. Frog Behavior under Microgravit)cPossibility of Motion Sickness in Amphibia, Proceedings of rhe 9rh ISAS Space UtilizationSymposium:1 12-1 14, 1992. 14. Wassersug, R.J., Pronych, S., Izumi-Kurotani, A., Fejtek, M. The Behavioral Responses of Vertebrates to Microgravity: A Comparative Approach. Proceedings of Space Bound ’93:73-74, 1993. 15. McNally, W.J., Tait, J. Ablation experiments on the labyrinth of the frog. American Journal of Physiology, 75:155179, 1925. 16. Gray, J., Lissrnan, H.W. The effect of labyrinthectomy on the coordination of limb movements in the toad. Journal of Experimental Biology, 24:34-40, 1947. 17. Suzuki, M., Harada, Y., Omura, R., Fujii, M., Hirokawa, H., Mori, N. Effect ofselectivevestibular neurectomy on frog motor function. Otologia Fukuoka, 36(2):25%263, 1990 (Japanese).
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18. Ikeda, T., Sekitani,T., Kido,T., Kanaya, K., Tahara, T., Hara, H. Study on EquilibriumoftheSmall Animal Using Drop Shafi-Usability of the Frog for the Vestibular Neurectomy and Experimental maintenance-Proceedings of the Ninth ISAS Space Utilization Symposium: 9W02, 1993. 19. Naitoh, T., Wassersug, R.J., Leslie, R.A. The physiology, morphology, and ontogeny of emetic behavior in anuran amphibians. Physiological Zoology, 62(3):81W 4 3 , 1989. 20. Akiyama, T. Interview one month after the recovery, 1991. 21. Akiyama, T. Report on the third day in space, 1990. 22. Kashima, I., Nishimura, K., Okamoto, Y., Kanno, M. Image analysis of bone changes in Hyla japonica exposed to microgravity on the MIR orbital station. Biological Sciences in Space, S(3):190-193, 1991. 23. Ohira, Y., Wakatsuki, T., Saito, K., Kuroda. A., Tanaka, H., Izumi-Kurotani, A,, Yamashita, M. Responses of P-adrenoreceptors in frog and rat hind limb muscles to gravitational unloading and/or creatine depletion. Biological Sciences in Space, 5(3):194-199, 1991. 24. Yamazaki, H., Kita, F., Ikuma, K., Ohnaka, H., Koike, K., Takahashi, S., Shiraishi, A., Ohashi. S. Effects of gravity and oriental medicine, tochu (Eucommia ulmoides Oliver) leaves, on tree frog Hylajaponica. .Biological Sciences in Space, 5(3):202-207, 1991. 25. Suzuki, M., Harada, Y., Takumida, M., Sekitani, T., Tahara, T., Kanaya, K. Vestibular sensory Epithelia of the tree Frog returned from space. Biological Sciences in Space, 5(3):20%21 1, 199 I. 26. Feuilloley, M., Yon, L., Kikuyama, S., Okuno, M., Kawamura, K., Gutkowska, J., Vaudry, H. Effect of space flight on the distribution of atrial natriuretic peptide (ANP)-like immunoreactivity in the heart of the tree frog, Hyla japonica:-Preliminary Report-. Biological Sciences in Space, 5(3):215-2 17, 1991. 27. Feuilloley, M., Yon, L., Kawamura, K., Kikuyama, S., Gutkowska, J.. Vaudry, H. lmmunochemical Localization of atrial natriuretic factor (ANF)-like peptides in the brain and heart of the tree frog, Hyla japonica: -Effect of weightlessness on the distribution of immunoreactive neurons and cardiocytes. Journal of Comparative Neurology, 330:32-47, 1993. 29. FRIS Experiment Group, Report ofFrog Experiment OnboardSpace Station MIR, Space Utilization Research Center, Institute of Space and Astronautical Science. Kanagawa. 199 1 (Japanese).
FROG EXPERIMENT ONBOARD SPACE STATION MIR Akem i IzumLKurotani. Yosh ihiro Mogami. Makoto Okuno. and Masamichi Yamashita I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Experimental System and Operation . . . . . . . . . . . . . . . . . . . . . . A . TreeFrog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Transportation of Frogs to Mir . . . . . . . . . . . . . . . . . . . . . . . C . Experiment in Orbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Recovery of Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Ground Control Experiment . . . . . . . . . . . . . . . . . . . . . . . . 111. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Experimentinorbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Control Experierntn on Ground . . . . . . . . . . . . . . . . . . . . . . C. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Space Biology and Medicine Volume 6. pages 193-211 Copyright 0 1997 by JAI Press Inc All rights of reproduction in any form reserved ISBN: 0-7623-0147-3
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1. INTRODUCTION Living creatures on earth have evolved in the earthly environment from birth. Gravity is one of the important parameters in the environment. Behavior of various animals on this planet is influenced by gravity. Some space experiments have been aimed at the study of the role of gravity in animal behavior, for instance in the construction of beeswax combs in a colony of honey bees,' in the mating behavior of parasitic wasps,' in the web-building behavior of garden cross spiders? in the swimmingbehavior ofjelly fish' and killifi~h,'.~ in the swimming behavior of larva and adult of African clawed frog? and in the behavior of space-hatched Japanese
quaii.5
In December 1990, we sent six Japanese tree frogs to the Russian space station Mir to investigate their posture and behavior in microgravity. Tree frogs present several types ofbehavior, i.e., walking,climbing,swimmingandjumping. Principal objective of this space experiment was to study how the frogs would respond to the condition of weightlessness. When a frog is briefly exposed to microgravity in free fall (1-2 seconds) or parabolic flight (about 20 seconds)? they arch their back and stretch out their four limbs. Spaceflight can greatly extend the duration of exposure to microgravity, in this mission to 8 days. In this experiment we observed the posture and behavior of Japanese tree frogs during a prolonged stay in the microgravity environment. Questions studied were, for example: When frogs try to change their position, do they select any original mode of behavior in microgravity?; Can they create a new way of locomotion? When an external stimulus is presented to the animals in orbit, is the stimulus strength changed from that observed on earth?
II. EXPERIMENTAL SYSTEM AND OPERATION A. Tree Frog
The Japanese tree frog, Hylujuponicu,is an arboreal animal, i.e., it normally lives on leaves and twigs of small grasses or shrubs. During the breeding season it appears in or near the water, such as in a wet rice field. Its fingers have round adhesive discs and poorly developed webs.' From a group of about 400 tree frogs, caught in fields in the Kanto area of Japan, 100 healthy specimens were selected for the experiment. One of the criteria for selection was the ability of the animals to change their body color relatively rapidly (within 10 minutes; see section III).' Of these animals 18 were transported to the training site Star City, Russia, in advance and kept there. The remaining 82 animals were transported to the launch site, 10 days before launch, and were added to the advance group. The ability to change body color rapidly was rechecked at the launch site. The age of the animals ranged from 7-1 2 months, and their body weight from 3-1 u
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Table 1. Selection Criteria for Animals 1 . Absence of visible injuries
2. Health condition expressed by posture Frogs in poor health often crouch. 3. Normality of vestibular functions Optokinetic nystagmus: Head rotation was observed when the stage on which a frog sits turns on dorm-ventral axis with angular velocity of 20-45 degreedsec. Gravity response: Reorientation of head was observed when stage on which frog sits was tilted to 3 0 4 5 degrees from horizontal. Two direction (right-left and anterior-posterior axis) were tested. Righting response: Righting response was observed after a frog was made to lie on its back by an operator’s hand.
The day before launch 12 frogs (six males and six females) were selected from the group of 100 animals by means of the criteria listed in Table 1. The 12 frogs were divided into two groups, six flight animals and six ground control animals. All animals were starved during the 10-day period preceding launch. This was done to prevent clogging of the air vents of the Life Support Box with feces. In orbit the frogs were not fed routinely, but they could eat meal worms, which were brought to serve as food in the experiment on feeding behavior. The intestinal microflora of the Japanese tree frog was analyzed in advance to ensure that the safety requirements for the cosmonauts would be met.’
B. Transportation of Frogs to Mir A Life Support Box, shown in Figure 1, was used for transportation of the living specimens to Mir. It travelled on the Soyuz, which stayed in transfer orbit for two days before docking to Mir. The Life Support Box has a three-layered structure. The top layer has seven compartments: six for one flog each, and one for 30 meal worms (living larvae of Tenebrio obscurus) serving as food inflight. The frog compartment snugly fits a sitting frog and is lined with polyurethane foam, so as to protect the animal against the vibrations and shocks during launch. The foam is water-soaked to keep the animal from dehydrating. The compartment for meal worms is not lined with polyurethane foam and is kept dry. During transportation the frogs cannot approach the meal worms. Zeolite in the top layer absorbs odor and urine from the living specimens. Each frog was installed in its compartment no less than 7 hours before launch. The middle and bottom layers of the Life Support Box house a small air pump and air ducts. The small air pump is powered by a Ni-Cd battery. The air ducts are made of soaked polyphenol foam to keep the air moist for frogs. There are two air
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Figure 1. Life Support Box used for transportation of frogs on Soyuz, which stayed in transfer orbit for two days before docking to Mir.
inlets in the bottom layer and two outlets in the top layer. Membrane filters with sub-micron pores are present in the air inlets and outlets for the purpose of bioisolation. Fresh cabin air was drawn into the Life Support Box by the air pump for 2 days during transportation to Mir. The temperature in the box was passively kept at the level of the crew cabin. C. Experiment in Orbit
The Frog Observation System was used for the experiment in orbit in Mir. It is composed of a glove bag for the animals (Figure 2), a tool bag with two sets of experiment tools (Figure 3), a CCD camera and an 8 mm videorecorder. It was shipped to Mir on the unmanned Progress, about 2 months prior to the anival of the frogs. Of the two sets of experiment tools, one set was for use inside the glove bag, the other for use outside the glove bag. A complete list of the two sets of tools is presented in Table 2. Tools were kept in pockets of the tool bag to prevent their scattering in weightlessness. On the day after rendezvous of S o p and Mir (three days after launch = day L + 3) the frogs were transferred from the Life Support Box to the glove bag. First, the Life Support Box with the living specimens, and the tool band with experiment tools to be used inside the glove bag, were placed inside the glove bag. They were
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Figure 2. Glove bag for observation and handling of frogs in bioisolation. fixed on the inner face of the glove bag by Velcro tape. Second, the glove bag was sealed by a seal clip bar, and inflated by air released from a small air cylinder. A polyurethane foam on the tool band was soaked with water from a water reservoir tube to keep the inside moist. Then the frogs were set free in the glove bag for observation, and were kept in this bag during the entire mission. Frogs and experiment tools in the glove bag were handled through gloves. The health condition of the frogs (alive, weakened, or dead) was checked daily by visual inspection for a few minutes. From that point observation of the animals was started. The experiment with observation and recording of the frogs was performed twice, on days L+3 and L+5, in order to determine whether any form of adaptation occurred. The subjects of observations are listed in Table 3. On the experiment days the behavior of the frogs was recorded with a small CCD camera or 8-mm VTR camera by a Japanese mission operator or a Russian cosmonaut.
D. Recovery of Frogs AFrog Recovery Box (Figure 4) was installed in the tool band, which was placed inside the glove bag at the beginning of the experiment. On the last day of the mission (day L+8) an appropriate volume of water was poured on a calcium peroxide tablet in the Frog Recovery Box to generate suficient oxygen gas for life support during the recovery phase. The polyphenol foam covering the interior
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Table 2. List of Tools a. Tools used outside Glove bag Name of Tool
Quantity
Seal clip bar Hand-powered Air Pump Air cylinder Pressure regulator and valve
3
Audio cassette tape Supporting band
1
Camera holder Scissors Mending tape Inner gloves
1 1 1 1
Gloves in exchange
1
1 1
1
1
Use for Sealing of glove bag Air withdrawal from glove bag at end of expt. Inflating glove bag at start of expt. Regulation of releasing pressure of air from air cylinder Voice stimuli (not used) Fixing position of observation window to the cosmonaut Fixing CCD camera head on observation window Cutting off part of glove bag for frog recovery Mending of damage of glove bag (not used) Absorption of sweat of hands in gloves of glove bag (not used) Gloves of glove bag for back-up cosmonaut (not used)
b. Tools used inside Glove bag Name of Tool
Quantity
Water reservoir tube Air blower Suction pipette Tweezers
4
Scissors Wrench-driver Mending tape Vinegar bottle Imitative snake Imitative meal worm Imitative willow sprig Needle (in Needle holder) Paper for wipe Paper for white balance Burial bag Burial box Polyurethane foam Joint tube
1 1 1 1
Frog recovery box
2 1 1
1 1 1
1 3 1 5 1
1 1
1
Use for Container of water Positioning of frog (not used) Positioning of frog (not used) Handling of vinegar-immersed filter paper (not used). Mechanical stimuli (not used) Picking up meal worm General use Screwing off of fuse of LSB Mending of damage of gloves (not used) Experiment of chemical stimuli (not used) Experiment of visual stimuli (not used) Experiment of visual stimuli Experiment of visual stimuli Euthanasia (not used) Wipe of observation window (not used) Adjusting white balance (not used) Keeping carcass Keeping burial bag with carcass Humidifier Connecting between LSB and outlet of glove bag to use Zeolite in LSB as deodorizer for gas from glove bag Recovery of frogs
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Figure 3. Tool bags with tools used outside (a) and inside (b) glove bag.
surface of the frog compartment in the Frog Recovery Box was soaked with water to provide moisture for the animal. The six frogs were placed in the individual frog compartments. The Frog Recovery Box was removed from the glove bag without breaking bioisolation by the technique shown in Figure 5 . The box was placed in the entrance port of the glove bag. The port was parted by two seal clip bars, which were placed
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Table 3. Subjects of Observations of Frogs 1. Behavior and posture in microgravity 2. Response behavior to various external visual stimuli real food (a living meal worm) small moving object (imitation willow sprig with leaves, imitation worm or tweezers) large moving object (hand of operator) 3. Orientation behavior on rotating object (water reservoir tube) 4. Ability to change body color
between the box and the body of glove bag. After cutting the space between the two seal clip bars by a scissors, the Frog Recovery Box was isolated from the glove bag and encapsulated. Each cut end was wiped with a sterilizing cloth. The encapsulated Frog Recovery Box was returned to the ground in the Soyuz recovery capsule. E. Postflight Experiments Behavior and Vestibular Function
Observation and recording of behavior after recovery were performed in a hall of the airport closest to the landing site, as early as two hours after landing. The process of jumping, landing, walking, and climbing a wall by the frogs was observed and recorded. The vestibular function was also tested. Test items and procedures were the same as those at the launch site before launch (see Table 1). These observations were repeated again at 12 hrs after landing.
Figure 4. Frog Recovery Box used for recovery of frogs upon return to Earth.
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figure 5. Procedure for isolation of Frog Recovery Box from the glove bag.
Preparation of Organs and Tissues From 2.5 hours after landing, two spaceflight frogs and two ground-control frogs were dissected and their organs and tissues were fixed by a procedure suitable for later analysis. The outline of the entire experimental procedure is diagrammatically represented in Figure 6 .
F. Ground Control Experiment Ground-control frogs were kept for about 2.5 days in an identical Life Support Box as used for the transport of the flight animals. They were then released in an air-opened box (200W x 125D x 140H mm) at the same time as the flight frogs were released in the glove bag on the Mir station. Temperatureand humidity in the
R
N
Lafe Access
LSB
e3
LSB
@ 1st EXD.L+3 davs 2nd Ex'p. L+5 da'ys
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Recovery
FRB FRB
R+(-2.5 hrs)
Observation Disection
R+(-2 hn) Early Access
R+(-lh)
C CI l>l >
B
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-1
!9
FRB :Frog Recovery Box lday
Figure 6. Outline of operation of the Frog Experiment.
LSB : Life Supporl Box 2.5 days -4-
SOYUZ
?3b
e
LSB
LSB
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two containers did not exactly simulate those in the flight Life Support Box and the glove bag in the Mir station. The control frogs were then placed in a Frog Recovery Box identical to that used inflight in reserve, and kept there for about 1 day. Behavior and vestibular function were observed and recorded on days L+3 and L+5, and after return of the flight animals.
111. RESULTS AND DISCUSSION A. Experiment in Orbit
Recorded video tapes were handed to us ten days after return (day R+10). The Japanese mission operator was interviewed about one month after his return. From the tapes and the interview the following was learned. Behavior of Floating Frogs
Frogs floating in the air inflight showed a posture similar to that during the few seconds of microgravity in a parabolic flight or free They arched their back, inflated their abdomen, and extended the four limbs with opened fingers and toes (Figure 7). During floating, synchronous movements (like breaststroke swimming or scissors-kick) and asynchronousmovements of both hind limbs were observed sometimes. These movements lasted for such a short period that they could not provide a system of coordinated motility for locomotion. Floating frogs tried to catch whatever object one of their four limbs touched. The limbs pointed in a rather
Figure 7. Behavior of a frog floating in weightlessness. Arched back, inflated abdomen, extended limbs with fingers and toes opened and hind limbs extended in lateral position.
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lateral direction, although the direction was not stable. The typical posture during floating in microgravity resembled, except for the position of the limbs, the posture observed during jumping on the ground (Figure 8). It has been reported that at dawn arboreal sub-tropical forest frogs parachute from a canopy of rain forest to the ground.lo The parachuting frogs “move their front and hind limbs lateral to their bodies and spread their fingers and toes during aerial descent, adjusting limb position slightly during flight....... this lateral position of the limbs is quite different than that observed during the trajectoryphase of a regular jump where the forelimbs are anterior and lateral but the hind limbs remain extended posterior to the body.”” According to this description, the parachuting posture appears to bear a close resemblance to the floating posture in orbit. However, it is not known whether the Japanese tree frog makes a retarded descent like the subtropical forest frog. They may do it in a small way from shrubs or grasses. Further observation and analysis are required to clarify this point. This parachuting-like posture in orbit is not a common response to microgravity among frogs and toads. Recently we have been investigating the behavioral response of various frogs and toads to an abrupt decrease in gravity in free fall and parabolic These experiments suggest that the response can be related to
Figure 8. Jumping posture of frog postflight. Frog was made to jump from a platform (15 mm height) and land on polyurethane sheet. Recorded in darkness by 35-mm camera with multiple exposures in strobe light (0.1 sec. intervals). Frog extended its hind limbs backwards during the iump.
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their way of life.14In microgravity these frogs and toads, who are non-arboreal and move two-dimensionally on the ground surface (Rana rugosa, Rana nigromaculata, Xenopus laevis, Lepidobatrachus budgetti and Ceratophryssp.),tend to rotate around their rostral-caudal axis by means of scissor-like kicks. This long axis rotation is similar to their righting reflex when the animal is inverted in normal gravity, while arboreal or sub-arboreal frogs (Hyla japonica and Rachophorus schlegefii) often show a similar posture during parachuting. Behavior of Stationary Frogs
Though some frogsjumped off spontaneously,most of them did not move, stayed on a surface, or hid themselves under some object during the mission. Frogs frequently stayed on a moist polyurethane foam. Some frogs returned to a compartment in the Life Support Box in the glove bag. When a frog was sitting on a surface, like the glove bag, they frequently (four out of six frogs) assumed a particular posture. The neck was sharply bent backwards (at nearly right angle), the back was arched, the hind limbs were not folded completely, and the abdomen was pressed against the substrate (Figure 9). In this posture they would walk backwards. Posture in Microgravity
We have studied what this posture might express. The effects of selective neurectomy in the labyrinth on motor function has been investigated by several groups.15-'* This procedure might simulate the behavior experiment in microgravity, since the input of the gravity signal can be prevented by this neurectomy.
Figure 9. Typical posture of a frog sitting on surface of glove bag. Arched back, incompletely folded hind limbs, abdomen pressed against glove bag.
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Figure 10. Posture during emesis induced by CuSO4 solution. 10 pI CuSO4 solution (0.2 mg per g body weight) was administered by gastric cannula (photograph by Dr. T. Naitoh).
The posture and behavior observed after neurectomy do not directly explain the particular posture observed in Mir. Naitoh et aLt9 have reported that emetic behavior (vomiting and retching: emetic behavior in the absence of any regurgitation) can be induced in frogs by certain drugs. The particular posture in orbit resembles the posture during retching behavior induced by emetic drugs (Figure 10). We have subjected several species of frogs to parabolic flights in order to test whether frogs can suffer from motion sicknessby mechanical acceleration.12Some frogs showed signs of emetic behavior, like cyclical mouth opening and closing, blinking, and walking backwards during the flight, some frogs actually vomited after the flight. Thus we have concluded that frogs, including Hyla japonica, can suffer motion sickness from parabolic flight. Cyclical mouth opening and closing of the frogs was also observed by the mission operator in Mir.20The frogs showing the particular posture observed in Mir may thus have been in an emetic state, possibly due to motion sickness. Response Behavior
The body color of each frog was observed soon after their release from the Life Support Box into the glove bag. The color of the polyurethane foam lining of three compartments in the box was white, of the other three black. On the ground the frogs respond to the white color by a change of their body color to yellow-green within 10 min., to the black color by a change to green-black. According to the report of the mission operator,21one of the frogs from a white compartment turned dark soon after its release into the glove bag. This observation suggests that the
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body color change response to the background color may be inhibited in space. It was not clear whether the response was only delayed, since the mission operator made no later report about the body color. Another point is that the body color change depends on the light intensity, and it was not known whether the light intensity at the site of the experiment in Mir was sufficient to induce the body color change. The mission operator did not report about the colors of the frogs that had been transported in black compartments. When the mission operator brought his hand to the glove bag, he noticed an avoiding response as usually made from a large object. When the mission operator placed a meal worm in front of a frog sitting on the surface of the glove bag, the frog tried to catch it by a forefoot, but it failed to catch the worm. This failure seemed to be due to an instability of the footing when the frog put out its forefoot. When a small (95 mm length) pair of tweezers floated while rotating along its long axis, one frog looked toward it and oriented its body to it, however it could not approach the tweezers after jumping off. It seems that frogs in microgravity succeed in making a response behavior only when they keep a stable contact with a surface by means of the round adhesive discs on their fingers. Once they left the surface and floated, they could not control their movements sufficiently to respond to a stimulus. They could only approach a food object and eat it by keeping a steady contact with a surface. The hvo frogs, who were dissected after recovery, were found to have some food (meal worms?) in their stomach, although the mission operator could not observe eating behavior in orbit. Previously, it has been reported that space-hatchedJapanese quails could not approach their food without the help of a c~smonaut.~ Quail chicks might not be able to hold on to the substrate while moving toward their food. When a frog rode on a rotating water reservoir tube (220 mm length, 32-50 mm diameter), it walked along the circumference toward the opposite direction. The orientation behavior observed on the ground was also shown in orbit, however it is not clear whether the frogs oriented themselves by a visual standard, or by angular acceleration sensing, or both. Adaptation to Microgravity
After a frog jumped off in orbit, it would try to land or perch on some object, but often failed to do so. The frequency of failure to land or perch after ajump decreased with time: from an average of 2.7 random contacts with an object or surface per jump before proper landing on day L+3 to an average of 1.4 contacts per jump before landing on day L+5. This finding suggests that frogs can adapt to microgravity in the matter of landing after a jump. However, the typical posture while sitting on a surface and the parachuting-like posture during floating were maintained until the end of the experiment. Adaptation to microgravity in the behavior of other animals has been reported. A cross spider could build a better web after 3 weeks in microgravity than in the
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beginning of its stay in orbit.2 The frequency of looping behavior of killifish in microgravity gradually decreased after staying in orbit for several days.'33 B. Control Experiment on Ground
When the ground-control frogs were released from the Life Support Box or the Frog Recovery Box, their walking, climbing, swimming and jumping behavior and vestibular function (optokinetic nystagmus, gravity response and righting response) were normal. C. Postflight Experiments Behavior of Recovered Frogs
All six frogs were recovered and returned alive after eight days of spaceflight. Upon the first observation of the animals, two hours after return (R+2 hrs.), all six frogs walked slowly and climbed a wall toitteringly. After landing from a jump, the folding of the hind limbs was delayed. Vestibular function did not appear clearly. Half an hour later (R+2.5 hrs.), their behavior and vestibular function began to return to normal. At R+12 hrs. behavior and vestibular function were normal again. Abnormal swimming behavior (looping) in Xenopw larvae has been reported to continue for 1-2 days after their recovery from a 7-day pacef flight.^ We cannot conclude that readaptation to 1-G condition in adult tree frogs was faster, since there are few reports about the behavior of adult frogs and toads after recovery from spaceflight. Analysis of Organs and Tissues
The organs and tissues of recovered and ground-control specimens were distributed to several research groups for a histological and biochemical analysis of the effect of an 8-day spaceflight. Their findings are summarized below. Kashima et al. observed the weakening of a vertebral bone.22The spongy bone in the caudal articulatio vertebra was less dense in the spaceflight animals than in the ground-controls.In a quantitative analysis by means of a bio-imaging analyzer they found 20% less calcium density in the posterior joint of the seventh vertebra of a spaceflight frog. Ohira and coworkers found a decrease in the P-adrenoreceptor activity, which is thought to correspond to mitochondria1biogenesis, in the gastrocnemiusof spaceflight frogs.23 Yamazaki and coworkers found a decreased collagen content in the skin and a lowered protein synthesis in the liver of spaceflight Suzuki and coworkers studied the morphology of the vestibular sensory epithelium by means of scanning electronmicroscopyand light micro~copy.~~ No changes
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were found in the sensory cilia of the utricular macula and the semicircular canal cristae in spaceflight frogs. Feuilloley and coworkers examined in heart and brain of spaceflight frogs the distribution of atrial natriuretic factor (ANF)-like peptides, which regulate water and electrolyte balance and blood pressure.26327 The density and distribution of the staining were identical in the hearts of control and spaceflight frogs. In the ground-control frogs ANF-positive cell bodies were found in the parium and striatum of the telencephalon, the lateral forebrain bundle of the diencephalon, and the nucleus reticularis isthmi in the mesencephalon. These were absent in the spaceflight frogs. Conversely, ANF-immunoreactivity was observed in the posterior nuclei of the posterolateralis thalamus in spaceflight frogs, but in the groundcontrol frogs these nuclei were scarcely stained. The authors suggested that prolonged exposure to microgravity affects biosynthesis and/or release of ANFrelated peptides in discrete regions of the frog brain.
IV. CONCLUSIONS AND SUMMARY Japanese tree frogs (Hylajaponica) showed unique postures and behavior during an 8-day flight on 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. Frogs 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. This 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 external 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 P-adrenoreceptor activity of the gastrocnemius was decreased. Skin collagen and liver protein synthesis were lowered. The distribution of the atrial natriuretic factor-like peptides in the brain was changed.
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ACKNOWLEDGMENTS We thank the persons i n Japan and Russia, whose efforts made the “Frog I n Space” project possible. A partial list of names is provided in reference 29. T h e extended studies on the postflight behavior of frogs and toads would not have been possible without the cooperation of Dr. R.J. Wassersug and Dr. T. Naitoh.
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