Fish mating experiment in space--what it aimed at and how it was prepared.

The code name 'MEDAKA' was given to the fish experiment in the IML-2 (the second International Microgravity Laboratory), a Space-shuttle mission (STS-65) carried out in July 1994. Medaka is the Japanese name for a small fresh-water fish, Oryzias latipes. This experiment titled 'Mating behavior of the fish Medaka and development of their eggs in space' aimed to present data for designing the future fish-culture in space. The Medaka experiment accomplished its objectives to the point of 100%. The fish mated, laid eggs in space, and these eggs developed normally to hatching (coming out as a baby fish) under microgravity. Its success totally depended on selection of the four fish sent to space. This paper describes the aims of the IML-2 Medaka fish experiment and how it was prepared, together with a brief report on what were achieved in space.     

Ten years after medaka fish mated and laid eggs in space and further preparation for the life-cycle experiment on ISS.

In the second International Microgravity Laboratory (IML-2/STS-65) mission in 1994, medaka fish (Oryzias latipes) performed their successful mating behavior in space for the first time among vertebrate animals. The eggs the fish laid in space developed normally, and hatched as fry (baby fish) in space. Those fry born in space had the same number of germ cells as the ground control fish, and these germ cells later developed to produce the offspring on the ground. Fry hatched in space did not exhibit any looping behavior regardless of their strain, visual acuity, etc. For fish, one of the most exciting experiments to be done abroad the International Space Station (ISS) is a realization of fish life cycles in microgravity. At present, fish are the most likely candidates to be the first vertebrate to live their life cycle in space. Research done in our laboratory for realizing the life-cycle experiment of medaka fish are also introduced.     

Development and swimming behavior of Medaka fry in a spaceflight aboard the Space Shuttle Columbia (STS-107)

A space experiment aimed at closely observing the development and swimming activity of medaka fry under microgravity was carried out as a part of the S*T*A*R*S Program, a space shuttle mission, in STS-107 in January 2003. Four eggs laid on earth in an artificially controlled environment were put in a container with a functionally closed ecological system and launched on the Space Shuttle Columbia. Each egg was held in place by a strip of Velcro in the container to be individually monitored by close-up CCD cameras. In the control experiment, four eggs prepared using the same experimental set-up remained on the ground. There was no appreciable difference in the time course of development between space- and ground-based embryos. In the ground experiment, embryos were observed to rotate in place enclosed with the egg membrane, whereas those in the flight unit did not rotate. One of the four eggs hatched on the 8th day after being launched into space. All four eggs hatched in the ground unit. The fry hatched in space was mostly motionless, but with occasional control of its posture with respect to references in the experimental chamber. The fry hatched on ground were observed to move actively, controlling their posture with respect to the gravity vector. These findings suggest that the absence of gravity affects the initiation process of motility of embryos and hatched fry.     

西藏双须叶须鱼早期发育特征

通过双须叶须鱼(Ptychobarbus dipogon Regan)早期发育特征研究, 旨在为该鱼的科学保护和合理开发提供技术支撑。结果显示: 双须叶须鱼卵径3.7—3.9 mm, 吸水后的卵径可达5.1—5.3 mm。在水温10℃左右的条件下, 经历336.02h孵化出膜。根据胚胎的外部形态特征可将胚胎发育分为准备卵裂阶段、卵裂阶段、囊胚阶段、原肠阶段、神经胚阶段、器官分化阶段、孵化阶段共7个阶段34个时期。初孵仔鱼全长12.4 mm, 第1天体色素出现, 胸鳍上翘, 鳃盖骨出现, 下颌原基出现; 第2天鳃弓原基出现; 第3天消化道出现, 肝胰脏原基出现; 第4天鳃耙出现, 体表色素细胞带出现; 第5天口凹形成, 鳃丝形成; 第6天胸鳍褶, 背鳍褶, 腹鳍褶出现; 第7天鼻凹出现, 星芒状色素团出现; 第9天鳔前原基出现; 第11天尾鳍鳍条开始出现, 胸鳍开始颤动; 第13天鳔1室出现, 半规管形成; 第17天背鳍分化出来; 第21天腹部鳍褶变大, 舌颌骨清晰可见; 第28天脾脏出现; 第33天出现腹鳍鳍条; 第34天鳞片出现; 第85天稚鱼的形态与成鱼无异。双须叶须鱼是已报道裂腹鱼类卵径最大, 较四大家鱼卵周隙小, 是对高原隆起所导致的高寒自然环境的一种适应。     

Embryogenesis and swimming behavior of Medaka fish in JUSTSAP STARS Program (STS-107).

JUSTSAP (Japan-US Science, Technology and Space Application Program) Medaka fish experiment was carried out as a part of STARS (Space Technology and Research for Student) experiment, a space shuttle mission, STS-107 in January 2003. Four eggs laid on earth under artificially controlled environment were put in a closed ecological system, AHAB (Aquatic Habitat), and launched by Space Shuttle Columbia. For the control experiment, four eggs were put in the AHAB and remained on the ground. There was no remarkable difference in the time course of the development. In ground experiment embryos were observed to rotate in the egg membrane, whereas in flight unit they did not rotate. One egg hatched out on L (Launch) +8 days in flight unit. Four eggs hatched out in ground unit. Fry in flight unit was observed to face its back usually to the camera with little swimming movement. Fry in ground unit were observed to move actively and also to control their posture with respect to gravity vector.     

Induction of female-to-male sex reversal by high temperature treatment in Medaka, Oryzias latipes

Medaka, Oryzias latipes, has a firm XX-XY sex-determining system with the sex-determining gene, DMY, on the Y chromosome. However, previous studies have suggested that high water temperature might affect sex determination in Medaka. In the present study, the influence of high water temperature on sex reversal was examined. Fertilized eggs of two inbred strains of Medaka were developed at high water temperature (32 degrees C) until hatching. The hatched fry were kept at normal water temperatures (27 degrees C) until adulthood, and the phenotypic and genotypic sex was examined. As a result, 24% (N=105) and 50% (N=36) of XX fish developed a male phenotype in the Hd-rR and HNI inbred strains, respectively. These XX sex-reversed males had a normal testis and were fully fertile. On the other hand, all XY fish were male in the both strains. These results demonstrate that high water temperatures can induce XX sex reversal and that elevated water temperatures during the embryonic stage is a simple and useful method for getting XX males in Medaka.     

Explanations for a video version of the first vertebrate mating in space--a fish story.

In 1994, Japanese killifish, Medaka (Oryzias latipes) mated, laid eggs, and had babies in space. This was the first successful mating behavior carried out in space by vertebrate animals. Recently, the author has edited a video tape, which contains the video images of the fish mating, development of eggs, swimming behavior of baby fish in space, and also scenes of various preparatory experiments on the ground. The video tape is available, without any charge when requested to the author (K. Ijiri). This paper explains for each scene of the video tape.     

Histological and Transcriptomic Analysis of Adult Japanese Medaka Sampled Onboard the International Space Station

To understand how humans adapt to the space environment, many experiments can be conducted on astronauts as they work aboard the Space Shuttle or the International Space Station (ISS). We also need animal experiments that can apply to human models and help prevent or solve the health issues we face in space travel. The Japanese medaka (Oryzias latipes) is a suitable model fish for studying space adaptation as evidenced by adults of the species having mated successfully in space during 15 days of flight during the second International Microgravity Laboratory mission in 1994. The eggs laid by the fish developed normally and hatched as juveniles in space. In 2012, another space experiment (“Medaka Osteoclast”) was conducted. Six-week-old male and female Japanese medaka (Cab strain osteoblast transgenic fish) were maintained in the Aquatic Habitat system for two months in the ISS. Fish of the same strain and age were used as the ground controls. Six fish were fixed with paraformaldehyde or kept in RNA stabilization reagent (n = 4) and dissected for tissue sampling after being returned to the ground, so that several principal investigators working on the project could share samples. Histology indicated no significant changes except in the ovary. However, the RNA-seq analysis of 5345 genes from six tissues revealed highly tissue-specific space responsiveness after a two-month stay in the ISS. Similar responsiveness was observed among the brain and eye, ovary and testis, and the liver and intestine. Among these six tissues, the intestine showed the highest space response with 10 genes categorized as oxidation–reduction processes (gene ontogeny term GO:0055114), and the expression levels of choriogenin precursor genes were suppressed in the ovary. Eleven genes including klf9, klf13, odc1, hsp70 and hif3a were upregulated in more than four of the tissues examined, thus suggesting common immunoregulatory and stress responses during space adaptation.     

西藏巨须裂腹鱼早期发育特征

巨须裂腹鱼(Schizothorax macropogon)隶属裂腹鱼亚科, 裂腹鱼属, 是西藏特有经济鱼类, 因过度捕捞, 其种群数量和分布面积下降, 在2009年中国红色名录评为“濒危”等级。研究通过研究巨须裂腹鱼早期发育特征, 旨在为该鱼的科学养护提供技术支撑。结果表明: 巨须裂腹鱼受精卵直径3.0—3.2 mm, 遇水开始具有微黏性, 随后脱黏, 经过准备卵裂阶段、卵裂阶段、囊胚阶段、原肠胚阶段、神经胚阶段、器官分化阶段、 孵化阶段, 在水温10℃的条件下, 经过460.67h孵化出来。初孵仔鱼体长9.9—1.1 mm, 心率48—50次/min, 鳃盖骨清晰可见, 下颌原基、尾鳍下骨原基可见。第2天鼻凹出现; 第3天肝胰脏原基出现; 第4天鳃耙、肩带原基出现; 第6天仔鱼上下颌开始张合; 第7天心血管分化结束, 仔鱼开始进入混合营养期; 第14天鳔一室和体侧色素带形成; 第26天肋骨原基出现; 第35天鳔二室出现, 卵黄囊耗尽; 第63天背鳍分化结束; 第83天臀鳍分化结束。巨须裂腹鱼胚胎具有独特的发育时序: 体节的出现先于胚孔封闭, 是对高原环境的一种适应和进化。     

Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.     

Differences in egg size, shell thickness, pore density, pore diameter and water vapour conductance between first and second eggs of Snares Penguins Eudyptes robustus and their influence on hatching asynchrony

Brood reduction in birds is frequently induced by hatching asynchrony. Crested penguins (genus Eudyptes) are obligate brood reducers, but in contrast to most other birds, first‐laid eggs are considerably smaller in size than second‐laid eggs; furthermore, first‐laid eggs hatch after their siblings. The mechanisms underlying this reversal in size and hatching order remain unclear. In this study, we tested whether the second‐laid eggs of Snares Penguins Eudyptes robustus have a higher eggshell porosity allowing them to maintain a higher metabolic rate throughout incubation and to hatch before their first‐laid siblings. We investigated differences in egg size, shell thickness, pore density, pore diameter and water vapour conductance between first and second eggs within clutches and examined the influence of these shell characteristics on hatching asynchrony. First‐laid eggs of Snares Penguins were approximately 78% of the size of the larger second eggs. Second‐laid eggs had considerably thicker shells and more pores per cm² than first eggs, whereas pore diameter did not differ between eggs. Water vapour conductance was greater in second‐ (16.8 mg/day/torr) than in first‐laid eggs (14.9 mg/day/torr). The difference in water vapour conductance between first‐ and second‐laid eggs within clutches was related to hatching patterns. In nests where second eggs hatched before first‐laid eggs, second eggs had a considerably greater water conductance than their sibling, whereas in nests where both eggs hatched on the same day, the difference in water conductance between eggs was very small, and in a few nests where small first eggs hatched before their larger sibling, they had a greater water conductance than their larger second‐laid nestmate. Surprisingly few studies have investigated differences in shell characteristics between eggs within clutches and associated effects on hatching asynchrony. This study has demonstrated that such differences exist between eggs within clutches and that they can influence hatching patterns.     

Adaptive difference in daily timing of hatch in two locust species,Schistocerca gregaria and Locusta migratoria: The effects of thermocycles and phase polyphenism

This study examined the effects of temperature and phase polyphenism on egg hatching time in the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria. The two species exhibited differences and similarities in hatching behavior when exposed to different temperature conditions. In 12-h thermocycles of various temperatures, the S. gregaria eggs hatched during the cryoperiod (low temperature period), whereas L. migratoria eggs hatched during the thermoperiod (high temperature period). The eggs of both species hatched during the species-specific period of the thermoperiod in response to a temperature difference as small as 1 °C. Furthermore, the locusts adjusted hatching time to a new thermal environment that occurred shortly before the expected hatching time. In both species, the hatching of the eggs was synchronized to a specific time of the day, and two hatching peaks separated by approximately 1 day were observed at a constant temperature after the eggs were transferred from thermocycles 3 days before hatching. Eggs laid by gregarious females hatched earlier than those laid by solitarious females in S. gregaria but this difference was not observed in L. migratoria.     

Anserine and free amino acid contents in the egg and fry stages of developing rainbow trout (Oncorhynchus mykiss).

1. Anserine and free amino acid contents were examined in rainbow trout eggs, yolk sac and yolk-free fry before feeding. 2. Free amino acid levels showed little change in eggs and yolk sac and had a tendency to increase in yolk-free fry. 3. No anserine was detected in the eggs and yolk sac, but yolk-free fry significantly increased its concentration after hatching. This continuous increment of anserine was discussed in relation to buffering capacity of adult fish muscle.     

Oil droplets and yolk spheres during development of Medaka embryos

The sizes of oil droplets (globules) and the yolk sphere in the Medaka Oryzias latipes egg were measured in the developmental period from fertilization to hatching. Oil droplets coalesced with one another in the process of shifting toward the vegetal pole, and a single large oil droplet was finally located at the vegetal pole region in most eggs 2 days post-fertilization. The volume of the yolk sphere steeply decreased in the period from 2 to 8 days post-fertilization. The volume of oil droplets also declined linearly from 4 to 10 days post-fertilization. Lipid components exhibited no distinct change during embryogenesis. In order to verify whether oil droplets were required for development of Medaka embryos, oil droplets were artificially removed from the early developing embryos without the chorion (egg envelope). Naked embryos without the oil droplet developed normally to fry in the sterilized incubation medium and grew to the same mature fry as those grown from the control embryos.     

Effects of gravity on early embryogenesis [correction of embryogeneis] in Caenorhabditis elegans.

The embryonic development of the nematode Caenorhabditis elegans was examined under different gravitational conditions. The embryos after fertilization normally hatched under hypergravity (200 G) or 3D-clinorotation, whereas the number of eggs laid from an adult hermaphrodite decreased and their hatching rate was reduced under the hypergravity condition. The first cleavage plane in the 1-cell embryo was slid to some extent by re-orientation of liquid culture vessel, but the pattern and timing of cleavages were not affected.     

CLUTCH-SIZE, INCUBATION AND HATCHING SUCCESS IN THE HOUSE SPARROW AND TREE SPARROW PASSER SPP. AT OXFORD

Clutch-size, incubation and hatching success were studied in P. domesticus and P. montanus in 1961 and 1963–64 at Oxford. The most frequent clutch-size was four eggs in P. domesticus and five eggs in P. montanus . With one exception, colonies of P. domesticus showed no significant annual or local variations in its mean clutch-size; in P. montanus , however, there were significant annual variations in the mean clutch-size. Both species showed a seasonal increase followed by a decrease in their mean clutch-sizes.
Partial incubation occurred during the laying period of the clutch; sufficient incubation for continuous development of the embryo was apparently achieved when the last egg had been laid in clutches of two and three eggs in P. domesticus and in clutches of four eggs in P. montanus , but when the penultimate egg had been laid in larger clutches of both species. On average, hatching in P. domesticus occurred more or less synchronously in all eggs in clutches of two and three eggs, and in all eggs except the last one laid in larger clutches; the last egg in the larger clutches hatched up to a day after the others. It is suggested that this pattern of hatching was brought about by the pattern of incubation during the laying period.
P. domesticus had a lower hatching success than P. montanus , probably because fewer of its eggs were fertile.     

Change in the radiation responses of oogonia in the embryos and fry of the fish Oryzias latipes.

Embryos of the fish Oryzias latipes were irradiated with 1000 R of X-rays 1 day before hatching,and the post-irradiation change in the female germ-cell population was observed. Scarcely any reduction in the number of oogonia was observed, but their proliferation was inhibited. Repopulation occurred between 12 and 20 days after hatching. These responses were quite different from those of germ cells in the irradiated fry (Hamaguchi and Egami 1975). Embryos and/or fry were also exposed to 1000 R of X-rays 1 day before hatching and 0, 1, 2, and 3 days after hatching. A comparison of their responses suggested that the change in the radiation responses of oogonia is correlated with the initiation of meiosis.     

Water quality during egg incubation influences yolk-sac fry sodium kinetics in Atlantic salmon, Salmo salar L.: a possible mechanism of adaptation to acid waters

Atlantic salmon ( Salmo salar L.) fry hatched from eggs transferred from high-Na to low-Na water during the eyed stage of development had a significantly higher V_max and lower K_m (P <0.01) of the sodium uptake mechanism than fry hatched from eggs incubated entirely in low-Na or high-Na water.
Fry hatched from eggs transferred to acid, high aluminium water during the eyed stage of development had a similar V_max and K_m to fry hatched from eggs incubated entirely in high- or low-Na water. Eggs incubated continuously in acid, high aluminium (low-Na) water produced fry with significantly lower K_m and V_max values than fry hatched from eggs incubated continuously in low-Na water. Eggs and fry in acid, high aluminium water continually lost sodium and mortality was 100% at 5 5 M O degree-days (2–3 weeks after hatching).
The results are discussed with respect to the influence of perivitelline fluid ion activities in eggs in acid, high aluminium water on the kinetic characteristics of sodium uptake in yolk-sac fry. A possible mechanism for the long-term adaptation of teleosts in acidified natural waters is also proposed.     

Reproductive Strategies of the Insidious Fish Ectoparasite,Neobenedenia sp. (Capsalidae: Monogenea)

Fish monogeneans are lethal parasites in aquaculture. We provide the first experimental evidence that a notorious fish monogenean, Neobenedenia sp., can produce viable eggs in isolation for three consecutive generations. We infected individual, isolated, farmed barramundi, Lates calcarifer (Bloch) with a single oncomiracidium (larva) of the hermaphroditic monogenean Neobenedenia sp. Isolated parasites reached sexual maturity at day 10 post-hatch (24°C, 35‰) and laid ∼3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58±15 eggs) and peaked on day 15 (496±68 eggs) before gradually decreasing. Neobenedenia sp. exhibited egg laying and egg hatching rhythms. Parasites laid eggs continuously, but egg production increased in periods of darkness (64.3%), while the majority of oncomiracidia (81%) emerged from eggs in the first three hours of light. Eggs laid by isolated ‘parent’ parasites hatched and individual emerging oncomiracidia were used to infect more individual, isolated fish, with three consecutive, isolated, parasite generations (F1, F2 and F3) raised in the laboratory. Infection success and egg hatching success did not differ between generations. Our data show that one parasite, in the absence of a mate, presents a severe threat to captive fish populations.     

Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.