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.     

人工模拟产卵环境中青海湖裸鲤的繁殖行为

研究通过人工产卵环境模拟, 成功诱导青海湖裸鲤(Gymnocypris przewalskii)自然产卵受精, 通过标志跟踪和视频监控等方法对青海湖裸鲤的繁殖行为进行了研究。结果表明, 青海湖裸鲤的繁殖过程包含了追逐、伴游、潜尾、溅戏和交尾等典型行为。其中溅戏行为(单尾雌鱼或雄鱼背鳍露出水面并用尾部拍击水面)首次被观察确认, 常被误认为是交配行为。追逐、伴游、潜尾和溅戏等繁殖行为平均频次分别为: 5.7、14.7、9.3和3.5 次/d。通过采用不同性比重复配对实验发现, 在观测到的34次交配行为中, 32次(94.1%)均为1雌和1雄配对交配。研究还发现性成熟的雌性青海湖裸鲤为一次产卵类型, 短时间内多次交尾排卵, 雌雄交配(排卵授精)行为约每天3—9次, 每次排卵耗时约4—9s, 每次平均排卵量131粒(范围15—376), 每尾雌鱼可连续交配产卵3—7d。观测到34次的交配行为中, 31次(91.1%)均发生在夜间(23: 00—次日2: 00), 3次(8.9%)发生在白天(早晨6:00—7:00)。研究首次基于人工模拟产卵环境观察描述了青海湖裸鲤的繁殖行为特征, 可为青海湖裸鲤人工产卵场构建、自然繁殖监测和产卵场保护提供科学依据。     

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.     

Primordial germ cells in the embryos of Medaka fish.

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

Cannibalism and protective behavior of eggs in Arctic charr (Salvelinus alpinus)

From video recordings of spawning events, we quantified protective and cannibalistic behavior of Arctic charr occurring immediately after spawning. The number of fish cannibalizing on stray eggs was examined regarding (a) whether more than one male shed milt during the spawning event, that is, whether sperm competition occurred, (b) whether the sperm competition included few or many males, that is, the intensity of sperm competition, and (c) the density of fish at the spawning site. Response behavior toward egg cannibalism was also examined among females and dominant males in order to determine any parental investment toward protecting the eggs after spawning. Cannibalistic behavior was seen in almost 50% of the spawnings, and the multiple spawning events showed the highest numbers of fish cannibalizing on eggs. Both the number of males releasing milt and the number of fish approaching the spawning site were positively correlated with egg cannibalism. Sperm competition was, however, not a prerequisite for egg cannibalism. Although we also observed partial filial cannibalism, protective behavior of eggs was seen both among dominant males and females, suggesting that charr actually conduct parental care.     

Predominant maternal egg care and promiscuous mating system in the Japanese filefish,Rudarius ercodes (Monacanthidae)

Synopsis Reproductive behavior of the Japanese filefish, Rudarius ercodes, was studied at the rocky reef off Koinoura, northern Kyushu, Japan, between June and October 1989. Aggressive display was observed between males, but they were not territorial. Males had four types of courtship behavior: vibrating, tail bending, leaning and nuzzle. Spawning occurred early in the morning. A female and 1–3 male(s) mated together on brown algae. Each female spawned repeatedly with an interval of 6–12 days. Females cared for eggs and embryos from just after spawning until hatching, 2–4 days. Female egg care consisted of tending and guarding. Females tended eggs by blowing water on them and by fanning them with their pectoral fins. Females guarded eggs by driving away fish passing nearby. In some cases, males also guarded eggs by staying near the eggs and driving away conspecific males. Whether a male cares for eggs with a female seems to be affected by the form of mating (pair mating or single female-multiple male mating), and the probability of further reproduction after spawning. Dominant males showed a tendency to pair with a specific female intermittently over a two-month period. Mating, however, did not always occur between members of such pairs, and mates appeared to be inter-changeable with a promiscuous mating system.     

Female spawning strategy in Rhinogobius sp. OR: how do females deposit their eggs in the nest?

 We tried to elucidate how females of a paternal nest brooding goby Rhinogohius sp. OR deposit their eggs in a nest, using a marking technique for live eggs under laboratory conditions in which male somatic condition, nest space, and mating pattern (monogamous or bigamous) were controlled. Whether females rejected mating was independent of either male quality, such as body size and somatic condition, or nest space. In a situation in which two females were allowed to spawn sequentially with a male, however, females rejected mating at a higher rate when they were the first to spawn than when they were the second to spawn; this is because eggs from first females were more vulnerable to cannibalism by parental males and second females. Even when nest space was limited and thus was occupied by eggs from the first females, second females could deposit all their eggs in the nest by using the minute interspace of existing eggs. In the presence of the parental male, such a female seemed less likely to suffer a cost from increased egg mortality due to crowdedness, still holding the advantage of being the second spawner. Finally, we extrapolated the field breeding ecology of this fish from the laboratory data. It was suggested that a single monolayer brood of the same age usually consisted of eggs from multiple females and thus the mating pattern would be more polygynous than previously expected. Received: March 6, 2002 / Revised: July 11, 2002 / Accepted: August 14, 2002 RID="*" ID="*" Present address: Center for Marine Environmental Studies, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan (e-mail: nokuda@sci.ehime-u.ac.jp) Acknowledgments I am grateful to S. Sone and D. Takahashi for giving us useful information and to M. Inoue and H. Miyatake for their field assistance. This study was financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Correspondence to:Noboru Okuda     

Ovulation does not constrain egg parcel size in the simultaneous hermaphrodite Serranus subligarius

Among egg trading hermaphrodites, any factor which limits the number of eggs released by a female role hermaphrodite can potentially limit the mating success of the male role hermaphrodite fertilizing those eggs. This work examines the hypothesis that the timing of ovulation constrains the size of egg parcels and thereby limits male mating success in the egg parceling hermaphroditic fish Serranus subligarius. Two alternatives were evaluated: (1) Ovulation is a discrete event preceding spawning. It does not constrain the size of egg parcels and therefore does not limit mating success of male role partners. (2) Ovulation is an incremental process occurring throughout the spawning period. It limits the number of eggs available for release in each parcel and thereby limits mating success of the male role partner. Assessment of ovulation was conducted in a field stock of S. subligarius. Fish from size matched pairs were manually stripped at the onset of the spawning period or quarantined and sampled at the end of the spawning period. Fish sampled at either time point had the same number of eggs, suggesting that ovulation was a discrete event occurring at the onset of the spawning period. The diurnal cycle of ovulation was observed in naturally spawning hermaphrodites captured at intervals throughout the day. Ovulation began 2–4 h before spawning began. Some fish appeared to ovulate the entire day's clutch of eggs before spawning, while other fish released egg parcels before completing ovulation. I conclude that the pattern of ovulation is not uniform throughout the spawning stock. Because of the variability in timing of ovulation relative to parcel release, ovulation does not consistently limit the size of egg parcels and therefore is unlikely to be a physiological limit to male role mating success in S. subligarius hermaphrodites.     

The timing of spawning and egg production as constraints on male mating success in a simultaneously hermaphroditic fish

Synopsis The evolutionary stability of simultaneous hermaphroditism depends in part on the existence of constraints on the potential for male mating success. In the seabasses (Serranidae), several species of simultaneous hermaphrodites divide each day's clutch of eggs into parcels that are spawned sequentially and alternately with a partner. This behavior is thought to be one source of constraint on male mating success. A possible related source is the pattern of egg production. A study was therefore performed on the chalk bass,Serranus tortugarum, to examine this pattern. The results indicate that eggs are readied for spawning gradually over the course of the daily spawning period. The pattern of egg production acts jointly with spawning behavior in constraining male mating success. This pattern may be a pre-existing state to which the mating system has been adjusted, or it may have co-evolved with the mating system.     

Mechanism of vestibular adaptation of fish under microgravity.

In a space experiment, the adaptation of goldfish behavior during flight and readaptation after landing were investigated. Six goldfish (1 normal, 1 with otoliths removed on both sides, 4 with otoliths removed on one side) were flown in a fish package (F/P) of Aquatic Animal Experiment Unit (AAEU). The dorsal light responses (DLRs) of fish with otoliths removed were recorded after operation until launch and after landing. The behaviors of the fish were recorded with a video camera on Mission Elapsed Time (MET) Day-00, 02, 05, 08, 12. On MET Day-00, two fish with otoliths removed on one side showed flexion of body toward the operated side. These fish also showed rolling behavior toward the operated side. However, the body flexion disappeared on MET Day-05 or MET Day-08. No rolling behaviors were observed after that time. Five fish showed backward looping behaviors during the mission. Although the frequency of looping episodes decreased after MET Day-08, five fish still showed looping behavior on MET Day-12, that was the last day of video recording on orbit. In microgravity, visual system of fish did not seem to provide sufficient cues to prevent them from looping or rolling. After landing, no looping and rolling behavior was observed. However, the tilt angle of the DLR increased in the fish with otolith removed 5 month before launch but not in normal fish and those with otoliths removed 2 weeks before launch. These results suggest that the behavioral dysfunction and the adaptational process in space are dependent on vestibular inputs.     

Is egg carrying attractive? Mate choice in the golden egg bug (Coreidae,Heteroptera)

In several species of fish, females select males that are already guarding eggs in their nests. It is a matter of debate as to whether a female selects a good nest site for her offspring (natural selection) or a male for his attractiveness (sexual selection). The golden egg bug, Phyllomorpha laciniata Vill, resembles fish in the sense that mating males carry more eggs than single males, but in the bugs, female mate choice is decoupled from egg site choice. The sexual selection hypothesis predicts that if females select males using male egg load as a cue for male quality, they should not mate with a male when eggs are removed, regardless of his mating attempts. When individual females were enclosed with an egg-loaded male and an unloaded male, they mated equally often with both males, although the loaded males courted more. In addition, when only successful males were used, females mated equally often with the loaded male and the unloaded male irrespective of sex ratio. Male choice rather than female choice affected mating frequency when sex ratio was equal. Therefore, females do not select the male by the eggs he carries, but successful males may receive many eggs due to egg dumping by alien females while they mate or as a consequence of mate guarding.     

Effects of space environment on embryonic growth up to hatching of salamander eggs fertilized and developed during orbital flights.

In vertebrates, only few experiments have been performed in microgravity to study the embryonic development from fertilization. To date, these concern only amphibian and fish. We report here a study on the embryonic development of Pleurodeles waltl (urodele amphibian) eggs oviposited in microgravity. The experiment was performed twice on board the Mir space station and the data obtained included video recording and morphological, histological and immunocytological analyses. The data confirm that the microgravity conditions have effects during the embryonic period, particularly during cleavage and neurulation, inducing irregular segmentation and abnormal closure of the neural tube. Moreover, we observed several abnormalities hither to undescribed corresponding to cortical cytoplasm movements, a decrease of cell adhesion and a loss of cells. These abnormalities were temporary and subsequently reversible. The young larvae that hatched during the flight displayed normal morphology and swimming behavior after landing. The results obtained in the urodele Pleurodeles waltl are in accordance with those observed earlier in the anuran Xenopus laevis and in the fish Oryzias latipes.     

Females prefer mating males in the carmine triplefin,Axoclinus carminalis,a paternal brood-guarder

Synopsis This study investigates the role of male mating status in female choice patterns in the carmine triplefin, Axoclinus carminalis, a tripterygiid fish that exhibits paternal care. The distribution of daily reproductive activity is clumped, with many males receiving no mates and some receiving three or more. Females in this species do not prefer larger males, and characteristics of the oviposition site appear to have minimal effects on male mating success. When a female is removed from a male early in the daily spawning period, that male attracts fewer additional females for the remainder of the spawning period than does a control male. These changes in mating success are temporary, and do not affect mating success on subsequent days. A preference for mating males or males that are guarding eggs could provide asymmetric benefits for males to defend oviposition sites. This preference for males with eggs could be acting alone or with other factors such as high variance in oviposition site quality to favor the evolution of paternal care in fishes.     

Episodic electric discharges in the course of social interactions: an example of Asian clariid catfish

Function of weak electric discharges is conclusively proved only for two fish orders - Mormyriformes and Gymnotiformes. Every specimen of the two groups emits electric discharges continuously or quite regularly for location, orientation and communication. The function of weak episodic electric discharges in other groups of weakly electric fish - Rajiformes, Uranoscopidae and Siluriformes, remains the puzzle since Darwin. Recent experiments made it possible to expand the list of weakly electric fish with episodic discharges. The range of behavioral situations accompanied with electric emission has been expanded as well. For instance, Asian catfish, Clarias macrocephalus, emit episodic discharges while in aggressive and spawning behavior. Asian catfish females emit the special burst of electrical discharges as a part of mating ritual. This burst cannot serve as an invitation to spawning or synchronization of reproductive products release, because females emit it after the sperm ejection. If females would need males' help for eggs release, it could be suggested that discharges assist in their mutual efforts. Since the electric field strength near fish is higher than fish's non-specialized electrical sensitivity thresholds, other hypotheses are possible. For example, it could be suggested that electric fields would make sperm or eggs more active. To proceed in our conception about episodic discharges function, new hardware and software are needed.     

High levels of multiple paternity in a spermcast mating freshwater mussel

Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel (Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time.     

雪峰虫草寄主巨疖蝙蛾的生殖行为节律

巨疖蝙蛾Endoclita davidi Poujade是雪峰虫草菌Ophiocordyceps xuefengensis的重要寄主昆虫,对其生殖生物学的研究,可以为该虫的规模化人工饲养与雪峰虫草的培育提供依据.采用人工饲养结合室内红外摄影,研究分析巨疖蝙蛾的求偶、交尾和产卵行为节律.巨疖蝙蛾在羽化当天的暗期即可求偶、交尾和产卵.求偶行为通常在进入暗期的5 min内开始,并主要发生在进入暗期的15 min内(P＜0.05);随后的交尾行为主要发生在进入暗期的10～ 25 min内;产卵行为也主要发生在暗期.巨疖蝙蛾有多次交尾和产卵的习性,其中,2日龄的求偶率、交尾率及日产卵量均最大,分别达87.33％、61.09％和979粒/雌,且随日龄增加而逐渐降低.卵散产,总产卵量平均达4250粒/雌.提示巨疖蝙蛾羽化时即已性成熟,其生殖行为均发生在暗期,进入暗期可能是触发其生殖行为的重要影响因素.     

Observations of mating behavior of the manta ray,manta birostris, at the ogasawara islands, japan

On 11 July 1997, the mating behavior of wild manta rays,Manta birostris, was observed while skin diving off Chichijima. Ogasawara Islands, Japan, and recorded with 49 underwater photographs and about 20 minutes of video tape. The female manta ray involved was estimated as being approximately 5 m in dise width (DW) and the two males involved, approximately 4 m DW. Copulatory behavior of the two males appeared to be almost the same, copulation itself being of the abdomen-to-abdomen type. Initially, the males chased the female for 20–30 minutes, all animals swimming at approximately 10 km/h. Each copulation event occurred within one meter of the surface, during which time the participating male grasped the tip of the female's left pectoral fin with his mouth. The clasper was inserted for 90 seconds (Male No. 1) and 60 seconds (Male No. 2), respectively. The mating behavior sequence of the manta rays involved following five steps. 1.: Male chases behind the tail of the female, attempting (several times) to grasp the latter's pectoral fin (chasing behavior). 2.: Male bites the tip of the female's pectoral fin, before positioning itself against the latter's underside (biting behavior). 3.: Male inserts a clasper into the cloaca of the female (copulating behavior). 4.: Male removes the clasper from the cloaca of the female, but continues biting the latter's pectoral fin (post-copulating behavior). 5.: Male releases the pectoral fin of the female, setting her free (separating behavior).     

Changes in the behavior of the female short-tailed cricket,Anurogryllus muticus (De Geer) (Orthoptera: Gryllidae) following mating

InAnurogryllus muticus females, mating stimulates burrow construction, burrow provisioning, feeding, egg production, and egg-laying. Since mating often occurs before the ovaries are fully developed, the time span between mating and oviposition is used for increased food intake and the accumulation of nutrient reserves in the fatbody. Oviposition triggers maternal care of eggs and emerging hatchlings, and blocks egg consumption. Hatchling behavior is investigated. When hatchlings eat eggs, they prefer newly laid over older, embryonic eggs.     

Relatively large males lower reproductive success in female zebrafish

Females can adjust their reproductive effort in relation to their partner’s perceived fitness value. In zebrafish (Danio rerio), large males are typically preferred mating partners. However, females have been observed to reduce their reproductive output with exceptionally large males but it remains unknown whether it is due to sexual harassment or aggressive behavior to establish and maintain dominance. Here, we study the association between relative male size, sexual harassment and dominance behavior, female stress status (stress behaviors and whole-body cortisol concentration), and reproductive success during a 4 day spawning trial. We found female cortisol to correlate negatively with female body size and positively with female dominance behavior. However, male and female behavior as well as female cortisol level were not related to relative male size. Females mating with relatively large males produced more and most of their eggs during the first spawning day, while females with smaller males produced few eggs during the first day but then increased egg production. Despite females produced more eggs when mating with relatively larger males, their eggs had substantially lower fertilization rates compared to females mating with relatively smaller males. Hence, overall, the reproductive fitness was lowest when females mated with a relatively large male. These findings could help to explain the maintenance of male size variation under natural conditions.