Medaka vasa is required for migration but not survival of primordial germ cells

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcT4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F-0, 14d post-fertilization, dpf) were exposed via the water for 70d to 0 (control), 10, 50 and 250 mu g L-1 PFOS, followed by a further 30d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70d experiment. Embryos and larvae (F-1) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 mu g L-1 PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30d in clean water. The triiodothyronine (T-3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality. (c) 2008 Elsevier Ltd. All rights reserved.

Experimental studies on the effects of toxic Microcystis aeruginosa PCC7820 on the survival and reproduction of two freshwater rotifers Brachionus calyciflorus and Brachionus rubens

Blooms of Microcystis aeruginosa frequently occur in many eutrophic lakes in China, however, there is very little experimental study on the relationship between Microcystis and rotifers from Chinese waters. The effects of different concentrations of toxic M. aeruginosa PCC7820 on two common freshwater rotifers Brachionus calyciflorus and B. rubens were investigated in laboratory experiments. B. calyciflorus was able to utilize this strain of M. aeruginosa as a food source. However, M. aeruginosa suppressed the survival and reproduction of B. calyciflorus at the highest concentration (10(6) cells/ml) probably due to the inadequate nutrition. B. rubens was inhibited by toxic M. aeruginosa PCC7820 and the inhibition increased with the increasing Microcystis concentration. Our study indicates that the two rotifers have different sensitivities to toxic M. aeruginosa and that toxic cyanobacteria may affect zooplankton community structure by differentially inhibiting the different zooplankton taxa.

Effects of live food and formulated diets on survival, growth and protein content of first-feeding larvae of Plelteobagrus fulvidraco

In recent years, much progress has been made in the rearing of fish larvae fed only artificial diets. A preliminary study was made in an attempt to evaluate the effects of live food and formulated diets on survival, growth and body protein content of first-feeding larvae of Plelteobagrus fulvidraco. Three test diets varying in protein level were formulated: Feed 1 containing 45% protein, Feed 2 with 50% protein and Feed 3 with 55% protein. Larvae fed live food (newly hatched Artemia, unenriched) were the control. The experiment started 3 days post-hatch and lasted for 23 days. At the end of the 23-day trial, survival was best in the control group (65.6%) whereby the final body weight and specific growth rate (SGR) were significantly lower than those in the test feed groups. At the same time, coefficients of variation for SGR and final body weight in the test groups were significantly higher than those in the control. Whole body protein content in all treatments showed a similar tendency during development: significantly higher 3 days post-hatch, then decreasing significantly, and then increasing unstatistically 10 days post-hatch. All results suggest that live food is still better for first-feeding larvae of P. fulvidraco, since live food leads to healthier larvae growth.

Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro

Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis in vitro and to develop approaches for germ-line transmission.

Effects of salinity on food intake, growth, and survival of pufferfish (Fugu obscurus)

The food intake, growth, food conversion ratio and survival of yearling pufferfish, Fugu obscurus Abe, were investigated under different water salinity conditions over a 54-day period. Within the salinity regimes of 0 (freshwater), 8, 18, and 35parts per thousand, the food intake levels were 0.97%, 1.43%, 1.19% and 1.01%, respectively; food conversion ratios were 1.31, 1.93, 1.61 and 1.36, respectively; and specific growth rates were 0.41%, 1.15%, 0.84%, and 0.35%, respectively. The three data series were reduced with increasing salinity. However, the survival rates did not show the same tendencies, which were 80%, 100%, 100%, and 67%, respectively. There were significant differences among the treatments. In conclusion, the yearling pufferfish optimum culture salinity condition was about 8parts per thousand.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural. observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

Survival, growth, sex ratio, and maturity of the Chinese mitten crab (Eriocheir sinensis) reared in a Chinese pond

Aspects of the biology of pond-cultured Chinese mitten crab (Eriocheir sinensis H. Milne-Edwards) were studied from June to November 1993. The survival rate of the population was estimated at 18.6%, and there was no significant difference between sexes in growth (t-test, P > 0.05). As the crabs grew from 7.3 to 33.8 mm in mean carapace length, seven molts were observed for the population. The intermolt period ranged from seven to 22 days and lengthened with increased size. Sex ratio at each sampling time did not differ significantly from 1:1 (Chi-square test, P > 0.05). Female crabs presumably required about eleven postlarval molts to reach sexually mature size, which was 34.1 +/- 3.9 (SD) mm. in carapace length in this study.

Independent adaptation to riverine habitats allowed survival of ancient cetacean lineages

The four species of "river dolphins" are associated with six separate great river systems on three subcontinents and have been grouped for more than a century into a single taxon based on their similar appearance. However, several morphologists recently questioned the monophyly of that group. By using phylogenetic analyses of nucleotide sequences from three mitochondrial and two nuclear genes, we demonstrate with statistical significance that extant river dolphins are not monophyletic and suggest that they are relict species whose adaptation to riverine habitats incidentally insured their survival against major environmental changes in the marine ecosystem or the emergence of Delphinidae.

STUDY ON THE MODE OF PRIMARY CONTROL IN THE EGG OF GYNOGENETIC CRUCIAN CARP FOR INHIBITING THE DEVELOPMENT OF 2 TYPES OF SPERM NUCLEI

By comparing the different developmental characteristics of two types of sperm nuclei which were from gynogenetic fish (crucian carp) and amphimictic fishes (red carp, red goldfish and sex-reversal red carp) respectively in the eggs of gynogenetic crucian carp, it was preliminarily revealed that there existed selective inhibiting actions of the primary control in the eggs of crucian carp for inhibiting the development of the two types of sperm nuclei. To homologous sperms, the primary control showed weak effect, thus leading to the decondensation of homologous sperm nuclei at different degrees in the eggs of crucian carp. But to heterologous sperms, the primary control showed strong effects, resulting in the total inhibition of the development of heterologous sperm nuclei. Moreover, our experimental results also showed that the different developmental behavior of the two types of sperm nuclei might have a great relationship to the changes of the sex ratio in the population of gynogenetic crucian carp. The infiltration of "the genetic materials in sperm nuclei" into the female nucleus at random might play an important role in male emergence in the naturally gynogenetic population of crucian carp.

镇海棘螈繁殖群体生态和遗传结构研究

genetics, such as: population size, reproduction, mating system, growth, development,genetic structure and systematics status; The main results are presented below: The seasonal variation of the operational sexual ratio of this animal was found in the field and the ration always bias the female in the breeding season. Aiming at this character and considering the distance of time and space of both sexual habitat in breeding season, we census female population first by toe-clipping mark-recapture method, then estimated the population size with the definitive sexual ratio. Up to now, this species was found only at the Beilun district of the Ningbo City. The population size of the Ruiyan Temple Forest Park approximates to 369. The status of this population is extremely endangered, so besides protecting this population at the original locality, we also suggested to breed the salamander in fenced locality and to hatch embryos artificially, and send metamorphosed juveniles back to nature. We can transfer some individuals to other similar habitats or breed them under artificial conditions for saving this species from extinction. The early developmental stage of the Chinhai salamander is the same as its relative species, E. andersoni. Their balanceres are poorly developed and disappear very early. Temperature and moisture significantly influence the embryonic development of the Chinhai salamander. The embryonic stage is approx. 29 days under room temperature. The hatchling grows in a logarithmic curve. The larvae stage in water is approx. 58- 88 days. Many factors influence the nomal development, including two aspects of internal and external. Due to these factors, the effective protected measures were presented in detail. The breeding migration of E. chinhaiensis takes place at late March～late April every year. This salamander's hatching rate is high, but the rate of hatchling migrating into water is low. The average effectiveness of all the nest sites is 36.7%. The maternal self-conservation was contrary to the reproductive success of the egg-laying strategy. In the strategy of egg-laying behavior, the first factor selected by the female was its self-conservation, the second is embryonic survival rate, and the last is rate of hatchling survival rate. The oviposition selection is significant for the survival of the larvae. Based on the analysis of the evolutionary process of reproductive behaviors nad egg-laying site selections of all genera of the family Salamandridae, we deduced that perhaps Echinotriton is a transitional type in the evolutionary process from water to land. Due to its location in the adaptive stage in the terrestrial evolution, Echinotriton chinhaiensis's terrestrial nest may be one of important reason that causes this species to be endangered. The genetic deversity analysis shows that although the population size of the Chinhai salamander is quite small compared to other Chinese salamandrid species, the genetic diversity of this population is not reduce remarkably. We explain this phenomena with the polygamy mating system of this species. The result of 4 families' parenthood determinations shows that the parenhood determination can be taken without any paternal information. The "children" of every female include rich genetic information from at least two "fathers". It implies that female Chinhai salamander mates more than once with different males in a breeding season. The molecular evidence, the behavioral observation evidences and the sperm evidence in the female cloaca proved that this species has a polygamy mating system. The kin recognition in the mating of adult salamander was first discussed. The taxonomic status and phylogenetic relationships of 12 species representing 6 genera in the family Salamandridae were studied using DNA fingerprinting. The results showed that the DNA fingerprinting. The results showed that the DNA fingerprinting patterns demonstrated rich genetic diversity and species diversity, and also revealed the taxonomic status and phylogenetic relationshipes of higher taxa to a certain extent. The results are highly consistent with those obtained from the studies based on the morphology, ecology, cytology and molecular biology. The compreshensive analysis indicate that Tylototrition hainanensis and T. wenxianensis should be valid species; Echinotriton should be a valid genus;Tylotortriton is a natural cluster; Tylotortriton asperrimus should be put in Tylototrition rather than in Echinotriton, Hypselotriton and Allomestriton are synonyms of Cynops and Paramesotriton, respectively. There are three main groups in Chinese salamandride: Cynops, Paramesotriton and Pachytrition from the first group, the species of the Tylototriton from the second, and E. chinhaiensis composes the third.