Ontogeny of the Lymphoid Organs of Japanese Flounder, Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells wereobserved within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.     

Ontogeny of the immune system in rock bream Oplegnathus fasciatus

Histogenesis of the immune system and specific activity of superoxide dismutase(SOD) were studied in rock bream Oplegnathus fasciatus from fertilization to 50 days after hatching(DAH).The pronephric tubule primordium developed in the embryo,14 h 30 min post fertilization.The spleen anlage was observed between the swim bladder and the intestine at 5 DAH,and the thymus was formed as a paired structure under the pharyngeal epithelium above the gill arch at 10 DAH.The order of the immune organs becoming lymphoid was the pronephric kidney(10 DAH),thymus(15 DAH) and spleen(21 DAH).As the embryo developed,the specific activity of SOD gradually increased until hatching,but subsequently SOD activity continuously decreased to a minimum at 14 DAH.After the spleen became lymphoid,the specific activity of SOD was relatively stable.It is suggested that the immaturity of the lymphoid organs and low specific activity of SOD was the cause of the high mortality of fingerlings 12 to 16 DAH.     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Species identification and phylogenetic analysis of genus Nassarius(Nassariidae)based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.     

Isolation and Characterization of Pathogenic Listonella anguillarum of Diseased Haft-Smooth Tongue Sole （Cynoglossus semilaevis Günther）

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among 14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 〈1.0×10^5 colony forming units （CFU）mL^-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillantm with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay （IFA） was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.     

Molecular Characterization and Expression Analysis of SKIV Infection of Interferon-Induced Protein with Tetratricopeptide Repeats 1(IFIT1) in Epinephelus lanceolatus

Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.     

The Clam Cyclina sinensis (Gmelin) Phylogeography Study with 28S rRNA Gene and Potential of Nuclear rRNA Genes in Genetic Assessments of Molluscs

Intraspecific diversity of molluscan species is usually studied based on maternally inherited mitochondrial DNA,from which only part of the evolutionary history can be reflected.Some nuclear ribosomal RNA genes such as 28S rRNA represent poten-tial candidates that can be easily applied in phylogeography because of lacking intraindividual variation.However,considering their low polymorphism,genetic appraisals on whether and how they can be used in population studies are necessary.Here,we applied a short 28S rRNA to assess genetic patterns of the clam Cyclina sinensis along the coast of China and compared the results with a for-mer study based on COI and ITS-1 analyses.The results revealed the 28S rRNA data set was characterized by an extremely low level of variation,with only seven haplotypes defined for 93 individuals.Haplotype and nucleotide diversity for each population was al-most the lowest when compared with the other two markers.However,the distribution of two dominant haplotypes showed clear geo-graphic patterns,and significant population differentiation was revealed between the East China Sea and the South China Sea.These patterns were highly concordant with findings of the former study that populations of C.sinensis were historically separated by land bridges among sea basins.Our study suggested that although the nuclear rRNAs have shortcomings such as low variation,they have advantages including lack of intraindividual variation and high amplification rates.Applying rRNA genes can enrich the toolbox of nuclear markers in molluscan phylogeographic studies.     

Distribution and sources of polycyclic aromatic, hydrocarbons in main aquacultural areas in Guangdong, China

The environmental quality status of Daya Bay (22.56–22.77°N, 114.51–114.73°E), a main aquaculture area in Guangdong of China, was investigated using 16 polycyclic aromatic hydrocarbon (PAH) sediment samples of the bay. The total concentrations of 16 PAHs varied from 115 to 1 134 ng/g dry weight. The PAH composition pattern in sediments suggest dominance of 4-ring PAHs in Sites 2 and 4, and the ratio of certain related PAHs indicated important pyrolytic and petrogenic sources. The results enhance the understanding of current contamination levels and make a better assessment of likely impacts of organic contamination on ecosystems and the sustainability of local aquaculture in the area especially after the establishment of the Nuclear Power Station by the bay.     

DISTRIBUTION OF DISSOLVED AND PARTICULATE TRACE METALS IN ANOXIC SEAWATER, SAANICH INLET, B . C. CANADA

This paper discusses the spatial and seasonal distribution character of dissolved and paniculate trace metals in the anoxic seawater of Saanich Inlet, B.C., Canada. The study showed that concentration of dissolved and paniculate trace metals in anoxic seawater is closely related to (1) the concentration of H2S and the depth of the O2-H2S interface, (2) the exchange of seawater in Saanich Inlet with outside seawater, (3) biotic action, and (4) the flushing event. The study was based in part on the ratio between trace metals and nutrients. There was a steep change in the concentration of dissolved trace metals at the O2-H2S interface. The concentration of dissolved trace metals in the H2S-controlled zone was dependent on the H2S there. The suspended matter in Saanich Inlet comes from the Hero Strait seawater, phytoplankton production and resuspension of flushed-up sediments. The concentration of particulate trace metals was rather low in Saanich Inlet and tended to increase with depth. The total concentra     

A New κ-Carrageenase Cgk S from Marine Bacterium Shewanella sp. Kz7

A new κ-carrageenase gene cgk S was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224 bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase Cgk S was classified into the Glycoside Hydrolase family 16. The cgk S gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1 and a yield of 69%. Recombinant Cgk S was most active at 45℃ and p H 8.0. It was stable at p H 6.0–9.0 and below 30℃. The enzyme did not require Na Cl for activity, although its activity was enhanced by Na Cl. Cgk S degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Methods Used to Study Bacterial Diversity in the Marine Environment around Qingdao

Pollution has a considerable effect on biological communities, in terms of size and diversity of the populations. Yet, the precise consequences of human activity on microbial communities in the marine environment are poorly understood. Therefore, in an ongoing collaborative research programme between Heriot-Watt University and the Ocean University of Qingdao, bacteria were isolated in 1999 and 2000 from marine sediment, seawater, seaweed, fish and shellfish, taken from locations in Shandong Province adjacent to Qingdao. Sampling locations were comprised of industrial and aquacultural sites and a clean, control site. In order to analyse microbial diversity, a polyphasic approach was adopted for characterisation of these isolates, specifically through examination of key phenotypic traits, i.e. using Biolog GN MicroPlateTM profiles, bacterial whole cell protein profiles and 16S and 23S rRNA gene sequences. These techniques yielded complex taxonomic data, which were subjected to statistical and cluster analyses. The application of these methods to studies of microbial communities is discussed.     

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product.     

Molecular detection of microbial communities associated with Microcystis vs Synechococcus dominated waters in Tianjin,China

Harmful cyanobacterial blooms cause many ecological disasters worldwide. During the development of cyanobacterial blooms, the diversity and domination of cyanobacterial taxa are of a particular concern. In this study, the microbial community structure within a water system, such as in Yuqiao Reservoir and Haihe River in Tianjin City, China, was compared by using next-generation sequencing. A total of 5 001 operational taxonomic units were obtained and clustered from filtered 16 S rDNA V3–V4 region sequences. The cyanobacterial and microbial structures greatly differed in these two water areas. Microcystis was dominant in Yuqiao, whereas Synechococcus was dominant in Haihe. Proteobacteria species were dominant among all detected samples. The relative abundances of Bacteroidetes and Planctomycetes were higher in Yuqiao Reservoir than in Haihe River, whereas Firmicutes and Verrucomicrobia were relatively abundant in Haihe River. Further analyses indicated that the domination of both cyanobacteria was strongly related to several environmental factors, such as total nitrogen, total phosphorus, and dissolved oxygen, reflecting the role of trophic states in shaping the dominance of cyanobacterial taxa. The present study provided the example for Microcystis and Synechococcus dominance along a cyanobacterial bloom in north China. Applying highthroughput sequencing could offer a wide field of vision in analyzing microbial community structures.     

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.     

Sporo-pollen Assemblage and Paleoclimate Events in Shelf Area of the Southern Yellow Sea Since 1.5 ka B.P.

Based on the authors‘ 1986 to 1994 sporo-pollen assemblage analysis in the southern Yellow Sea area, data from 3 main cores were studied in combination with ^14C, palaeomagnetic and thermoluminescence data. The evolution of the paleoclimate environments in the southern Yellow Sea since 15ka B.P. was revealed that, in deglaciation of the last glacial period, the climate of late glaciation transformed into that of postglaciation, accompanied by a series of violent climate fluctuations. These evolution events happened in a global climate background and related to the geographic changes in eastern China. We distinguished three short-term cooling events and two warming events. Among them, the sporo-pollen assemblage of subzone A1 showed some cold climate features indicating that a cooling event occurred at about 15 - 14ka. B .P. in early deglaciation. This subzone corresponds to the Oldest Dryas. In subzone A3 , many drought-enduring herbal pollens and some few pollens of cold-resistant Picea, Abies, etc. were found, which indicated that a cooling event, with cold and arid climate, occurred at about 12- 11ka. B.P. in late deglaciation. This subzone corresponds to the Younger Dryas. The sporo-pollen assemblage of zone B showed warm and arid climate features in postglaciation. Although the assemblage of subzone B2 indicated a cold and arid climate environment, the development of flora in subzone B2 climate was less cold than that in A3 . Subzone B2 indicated a cooling event which occurred at about 9ka B.P. in early Holocene. Subzone A2, with some distinct differences from subzone A1 and A3 , indicated a warming event which occurred at 14 - 13ka. B.P. and should correspond to a warming fluctuation. The sporo-pollen assemblage of zone C showed features of warm-moist flora and climate, and indicated a warming event which universally occurred along the coast of eastern China at 8 - 3ka B.P. in middle Holocene, and its duration was longer than that of any climate events mentioned above. This period was climatic optimum and belonged to an altithermal period in postglaciation.