Seasonal pattern of reproduction of Hizikia fusiformis (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China

The maturation pattern of sexual reproduction in Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeaophyta) was examined in 2003 at Yunao Bay, Nanao Island, Shantou, China. Maturation began in mid-April (seawater temperature 19-21 degrees C), reached the peak in mid-May (maturation rate ca. 70%, and seawater temperature 23.5-25 degrees C) and finished in late-June (seawater temperature 27.5-30 degrees C). The Hizikia plants continued to gain the length from the beginning of maturation season to reach a maximum mean length of 34.8 cm in mid-May, after which the mean length was reduced drastically due to the senescence and rupture of the larger plants in size. The major portion of the mature plants belonged to the larger plants between April and May, but to the smaller ones in June. It is suggested that the plant must achieve a critical size before reproductive maturation occurred. There was a positive relationship between the number of receptacles (NR), as well as the reproductive allocation (RA), and the plant size of Hizikia population, with the recorded maximum values of NR and RA being 1220 and 64.3% respectively, for a single plant.

Upstream regulatory region of zebrafish lunatic fringe: Isolation and promoter analysis

Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish 1fng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish 1fng by blast search at the Ensembl genome database (http://www. ensembl.org) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced 1fngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous 1fngEGFP-transgenic line. The eggs from 1fngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of 1fng transcripts in the fertilized eggs. This reveals that zebrafish 1fng is a maternal factor existing in matured eggs, suggesting that fish somitogcnesis may be influenced by maternal factors.

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H (O)) and expected (H (E)) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.

Acrossocheilus spinifer, a new species of barred cyprinid fish from south China (Pisces : Teleostei)

Acrossocheilus spinifer sp. nov. is described from the river basins in Fujian Province and the Han Jiang basin in Guangdong Province, south China. It is one of the barred Acrossocheihis species sharing five or six vertical bars on the flanks, with each bar being two scales in width. Acrossocheilus spinifer sp. nov. is morphologically most similar to Acrossocheilus wenchowensis. but distinguished from it by: a colour pattern of all vertical bars extending ventrally to the second scale row below the lateral line, and having the second bar placed posterior to the base of the last simple dorsal-fin ray, a blunt snout; and a stout, last simple dorsal-fin ray. Acrossocheilus spinifer sp. nov. resembles Acrossocheilus kreyenbergii and Acrossocheilus stenotaeniatus with which it shares a stout, last simple dorsal-fin ray with a serrated posterior edge, but is separated from both species by having a narrow median interruption in the lower lip, a lower jaw fully covered by the lower lip when viewed ventrally and the second vertical bar placed posterior to the base of the last simple dorsal-fin ray. The absence of a longitudinal stripe extending along the lateral line on the flanks in males readily distinguishes A. spinifer sp. nov. from Four other species, namely Acrossocheilus fasciatus, Acrossocheilus paradoxus, Acrossocheilus parallens, and Acrossocheilus jishouensis. (c) 2006 The Authors Journal compilation (c) 2006 The Fisheries Society of the British Isles.

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Stocking models of Chinese mitten crab (Eriocheir japonica sinensis) in Yangtze lakes

The fanning of Chinese mitten crab, a quality aquatic product in China and neighbouring Asian countries, has been developing rapidly in China since last decade. It reached a total yield of 3.4 X 10(5) tonnes in 2002. Due to the successive over-stocking year after year, many lakes in the mid-lower Yangtze Basin, the main farming area, are under deterioration, leading to a reduction of crab yield and quality, and, subsequently, a loss of fanning profits. Aiming at a normal development of crab culture and the sustainable use of lakes, an annual investigation dealing with lake environmental factors in relation to stocked crab populations was carried out at 20 farms in 4 lakes. The results show that the submersed macrophyte biomass (B-Mac) is the key factor affecting annual crab yield (CY). Using the ratio of Secchi depth to mean depth (Z(SD)/Z(M)), an easily measured parameter closely correlated to BMac, as driving variable, 10 regression models of maximal crab yields were generated (r(2) ranging 0.49-0.81). Based on the theory of MSY (Maximum Sustainable Yield), in combination with body-weight (BW) and recapture rate (RR) of adult crabs, a general optimal stocking model was eventually formulated. All models are simple and easy to operate. Comments on their applications and prospects are given in brief. (c) 2006 Elsevier B.V. All rights reserved.

EST-based identification of genes expressed in the hypothalamus of male orange-spotted grouper (Epinephelus coioides)

The orange-spotted grouper, Epinephelus coioides, is an important marine aquaculture fish, but its large-scale aquaculture has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish. Hypothalamus-pituitary-gonad is an important endocrine axis in regulating reproduction and sex differentiation. To reveal the molecular mechanism of hypothalamic physiological functions, we performed he studies on identification of genes expressed in the hypothalamus of male orange-spotted grouper using EST and RT-PCR strategy. A total of 1006 ESTs were sequenced, and 402 (39.96%) clones were identified as known genes and 604 (60.04%) as unknown genes. The 402 clones of known gene products represent transcripts of 18 1 genes. Moreover, the expression patterns of 26 unknown genes were analyzed in various tissues, such as liver, kidney, spleen, fat, heart, muscle, pituitary, hypothalamus, telencephalon, cerebellum, midbrain, medulla oblongata, ovary and testes. Five different categories of expression patterns were observed from them. Several unknown ESTs, such as DN551996, DN551998, DN552082, and DN552070, were detected to be hypothalamus-specific, brains-specific, or hypothalamus and gonad-specific genes. Interestingly, DN551996, not only exhibiting expression differences between ovary and testis, but also showing sex-dependent differences in hypothalamus of grouper, might play significant role in grouper reproduction or sex inversion. Further functional studies on these genes will provide more information on molecule regulation mechanism of sex inversion in groupers. (c) 2006 Elsevier B.V All fights reserved.

Effects of the phytoplanktivorous silver carp (Hypophthalmichthys molitrixon) on plankton and the hepatotoxic microcystins in an enclosure experiment in a eutrophic lake, Lake Shichahal in Beijing

The responses of nutrients, water transparency, zooplankton, phytoplankton and microcystins to a gradient of silver carp biomass (0, 18, 55, 110 g/m(3)) were assessed using enclosures in Lake Shichahai (Beijing). Picophytoplankton biomass increased with increasing fish stocking density (r=0.64, p=0.09). Silver carp significantly depressed zooplankton biomass, and thus, zooplankton grazing was too low to control phytoplankton. Intracellular microcystin (MC) content in the enclosures was correlated only to Microcystis biomass in the present study. Microcystis spp. biomass and intracellular microcystins content were much higher in lake water than those of enclosures with and without stocking fish. Stocking of silver carp could be an appropriate in highly productive Lake Shichahai, which naturally lacks of large cladoceran zooplankton. A fish stocking density of 55 g/m(3) was most efficient at controlling Microcystis blooms and increasing water clarity. Mean extracellular MC concentration in the lake water was almost the same with that of the enclosures with fish. (c) 2006 Elsevier B.V. All rights reserved.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Effects of age and dietary protein level on digestive enzyme activity and gene expression of Pelteobagrus fulvidraco larvae

The present research studied the effects of age and dietary protein level on pepsin, trypsin and amylase activity and their mRNA level in Petteobagrus fulvidraco larvae from 3 to 26 days after hatch (DAH). Three DAH larvae were fed three isoenergetic diets, containing 42.8% (CP 43), 47.3% (CP 47) and 52.8% (CP 53) crude protein. Live food (newly hatched Artemia, unenriched) was included as a control. The effects of age on enzyme activity and mRNA were as follows: pepsin and trypsin activity in all treatment groups showed a significant (P < 0.05) increase at the beginning and decrease later although the timing of decrease was not the same among treatment groups and between the digestive enzymes. Pepsin and trypsin mRNA level followed the pattern of their respective enzyme changes. Age significantly affected amylase activity (P < 0.05) while age had no effect on amylase mRNA during the experimental period. The four diets significantly (P < 0.05) affected activity and mRNA level of pepsin and trypsin. Diets did not affect amylase activity or mRNA level. These results suggest that the effects of age on pepsin and trypsin gene expressions are at the transcriptional level. Dietary protein level does affect pepsin and trypsin gene expression in the early life of P. fulvidraco. There were no transcriptional effects on amylase gene expression. (c) 2005 Elsevier B.V. All rights reserved.

Reproductive seasonality and maturity of male Neophocaena phocaenoides asiaeorientalis in captivity: A case study based on the hormone evidence

To provide hormone evidence on reproductive seasonality and maturity of the Yangtze finless porpoise, the authors monitored the monthly variations of serum reproductive hormones of a male Yangtze finless porpoise in captivity via radioimmunoassay from 1997 to 2003. It was demonstrated that the immature animal had a serum T level of 28-101ngdL -1 (6552ngdL -1 ), reached puberty with the serum T concentration ranging between 250 and 590ngdL -1 (390130ngdL -1 ), and attained reproductive maturity when the serum T level went beyond 1120180ngdL -1 in the breeding season. The body length growth of the animal showed a pattern similar to the serum T variations during its adolescent period from the age of 4 to 6 years. Moreover, the serum T concentration of the male porpoise exhibited significant seasonal variations, indicating that its breeding season may start as early as March and end as late as September.

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes : Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post-hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood-thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.

Seasonal variation of virioplankton in a eutrophic shallow lake

Lake Donghu is a typical eutrophic freshwater lake in which high abundance of planktonic viruses was recently revealed. In this study, seasonal variation of planktonic viruses were observed at three different trophic sites, hypertrophic, eutrophic, and mesotrophic regions, and the correlation between their abundances and other aquatic environmental components, such as bacterioplankton, chlorophyll a, burst size, pH, dissolved oxygen, and temperature, was analyzed for the period of an year. Virioplankton abundance detected by transmission electron microscope (TEM) ranged from 5.48 x 10(8) to 2.04 x 10(9) ml(-1) in all the sites throughout the study, and the high abundances and seasonal variations of planktonic viruses were related to the trophic status at the sampled sites in Lake Donghu. Their annual mean abundances were, the highest at the hypertrophic site (1.23x10(9) ml(-1)), medium at the eutrophic site (1.19x10(9) ml(-1)), and the lowest at the mesotrophic site (1.02x10(9) ml(-1)). The VBR (virus-to-bacteria ratio) values were high, ranging from 49 to 56 on average at the three sampled sites. The data suggested that the high viral abundance and high VBR values might be associated with high density of phytoplankton including algae and cyanobacteria in this eutrophic shallow lake, and that planktonic viruses are important members of freshwater ecosystems.