Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Comparative studies on photosynthesis and phosphate metabolism of Cylindrospermopsis raciborskii with Microcystis aeruginosa and Aphanizomenon flos-aquae

The physiological differences for three bloom-forming cyanobacteria (Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Aphanizomenon flos-aquae) were investigated. In comparison with M. aeruginosa and A. flos-aquae, C. raciborskii exhibited a significantly higher concentration of carotenoids, higher values in maximum photosynthesis rate (P-m), apparent photosynthetic efficieny (a), and maximum electron transport rate (ETRmax) during the growth period. In addition, higher extracellular alkaline phosphatase activities and lower light compensation point (I-c) were also detected in C raciborskii (p < 0.05, ANOVA). Therefore, it is suggested that the higher photosynthetic activities, more effective uptake and utilization to phosphate, and low light requirements might play important roles in the occurrence and invasive behavior of C. raciborskii. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2 alpha kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2 alpha kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2 alpha kinases might play similar roles and that PoPKR is the predominant kinase. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Mitigation of lake eutrophication: Loosen nitrogen control and focus on phosphorus abatement

w Traditionally, nitrogen control is generally considered an important component of reducing lake eutrophication and cyanobacteria blooms. However, this viewpoint is refuted recently by researchers in China and North America. In the present paper, the traditional viewpoint of nitrogen control is pointed out to lack a scientific basis: the N/P hypothesis is just a subjective assumption; bottle bioassay experiments fail to simulate the natural process of nitrogen fixation. Our multi-year comparative research in more than 40 Yangtze lakes indicates that phosphorus is the key factor determining phytoplankton growth regardless of nitrogen concentrations and that total phytoplankton biomass is determined by total phosphorus and not by total nitrogen concentrations. These results imply that, in the field, nitrogen control will not decrease phytoplankton biomass. This finding is supported by a long-term whole-lake experiment from North America. These outcomes can be generalized in terms that a reduction in nitrogen loading may not decrease the biomass of total phytoplankton as it can stimulate blooms of nitrogen-fixing cyanobacteria. To mitigate eutrophication, it is not nitrogen but phosphorus that should be reduced, unless nitrogen concentrations are too high to induce direct toxic impacts on human beings or other organisms. Finally, details are provided on how to reduce controls on nitrogen and how to mitigate eutrophication. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Seasonal Variations in Chlorophyll a Concentrations in Relation to Potentials of Sediment Phosphate Release by Different Mechanisms in a Large Chinese Shallow Eutrophic Lake (Lake Taihu)

The relationship between chlorophyll a and fractionation of sediment phosphorus, inorganic phosphate-solubilizing bacteria (IPB), and organic phosphate-mineralizing bacteria (OPB) was evaluated in a large Chinese shallow eutrophic lake (Lake Taihu) and its embayment (Wuli Bay). At the three study sites, the increase of chlorophyll a concentrations in April paralleled those of the iron bound phosphate accounting for major portion of sediment inorganic phosphate, and in June significantly higher OPB and IPB numbers (especially OPB) in sediment were main contributors to the peaks of chlorophyll a concentration. Even though IPB peaked from February to June, it should serve as an unimportant P source due to the irrelevancy with chlorophyll a and soluble reactive phosphorus (SRP). By contrast, at the other site in the embayment, the calcium-bound phosphate was predominant and solid, which was difficult to be released, and neither IPB nor OPB were detectable in the sediment, indicating weak potential for phosphorus release from the sediment, which was reflected in the small seasonal variation in SRP concentration in water column. Hence, the extents to which the three general mechanisms behind phosphate release from sediment (desorption of iron bound phosphate, solubilization by IPB and enzymatic hydrolysis by OPB) operated were different depending on seasons and sites in Lake Taihu, they may jointly drive phosphate release and accelerate the eutrophication processes.

CHARACTERISTICS OF FATTY ACID METHYL ESTERS (FAMEs) AND ENZYMATIC ACTIVITIES IN SEDIMENTS OF TWO EUTROPHIC LAKES

Enzymatic activities and fatty acid methyl esters (FAMEs) in the sediments of two eutrophic lakes in Wuhan city were investigated. The results showed phosphatase and dehydrogenase activities in the lotus zone and plant floating bed zone were significantly lower than those in other sites, and urease activity was the highest where microorganism agents were put in. Fatty acid group compositions indicated the predominance of aerobic bacteria in the surface sediments in shallow lakes. The ratios of FAMEs specific for bacteria and Gram-positive bacteria exibited significant differences between the two lakes. The results of trans to cis indicated that the microorganisms in Lake Yuehu could adapt themselves to environmental stress better. The enzymatic activities and FAMEs showed differences in different sites, indicating that ecological restoration measures and environmental conditions could affect lake sediment to some extent. But the monitoring, work would be done in series to exactly evaluate the effect of the remediation measures.

Two spawning stocks of icefish Neosalanx taihuensis revealed from annual reproductive cycle analyses

Neosalanx taihuensis were sampled from the Tian-e-zhou Oxbow from March 2006 through November 2007. Two separate spawning seasons were identified based on the annual reproductive cycles of the females, designated as the autumn-spawning season and the spring-spawning season. Lifespan of the offspring of the spring-spawning fish was 1 year, with them dying after the subsequent spring spawning. Autumn-spawned females seem to be the offspring of the spring-spawning fish, based on monthly changes in the length-frequency distributions. Spring-mature females had higher absolute fecundity, gonadosomatic index, and relative condition factor in 2007 than in 2006. Relative condition factor of the immature female offspring of spring-spawning fish was higher in 2007 than in 2006, portending a further increase in reproductive investment during the spring spawning of 2008. The increasing reproductive investment suggests that the population of N. taihuensis in the Tian-e-zhou Oxbow may be recovering from its recent decline.

Relationships between Testate Amoebae Assemblages (Protozoa) and Geographic Factors in Yunnan Plateau Lakes, China

Knowledge of the effect of geographic factors on the assemblages of protozoan testate amoebae is still limited, despite there having been a number of studies on this fauna. We applied statistical analyses to data on the distribution of testate amoebae from nine major lakes in the Yunnan Plateau, southwest China. Cluster analysis, based on community structure, separated the lakes into two groups - the oligotrophic/mesotrophic lakes and the hypercutrophic lakes - confirming the idea that the testate amoebae assemblages in lakes are closely related to the trophic status. Additionally, within the oligotrophic/mesotrophic lakes, there was distinct geographic clustering. Linear regression analysis and the Mantel test both revealed that similarity of species composition decreased with increasing geographic distance among the oligotrophic/mesotrophic lakes.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

Variables affecting melanoidins removal from molasses wastewater by coagulation/flocculation

Coagulation/flocculation process was applied in the polishing treatment of molasses wastewater on a bench-scale. Important operating variables, including coagulant type and dosage, solution pH, rapid mixing conditions as well as the type and dosage of polyeletrolytes were investigated based on the maximum removal efficiencies of chemical oxygen demand (COD) and color, residual turbidity and settling characteristics of flocs. HPSEC was utilized to evaluate the removal of molecular weight fractions of melanoidins-dominated organic compounds. Experimental results indicate that ferric chloride was the most effective among the conventional coagulants, achieving 89% COD and 98% color eliminations; while aluminum sulfate was the least effective, giving COD and color reductions of 66% and 86%, respectively. In addition to metal cations, counter-ions exert significant influence on the coagulation performance since Cl--based metal salts attained better removal efficiency than SO42--based ones at the optimal coagulant dosages. Coagulation of molasses effluent is a highly pH-dependent process, with better removal efficiency achieved at lower pH levels. Rapid mixing intensity, rather than rapid mixing time, has relatively strong influence on the settling characteristics of flocs formed. Lowering mixing intensity resulted in increasing settling rate but the accumulation of floating flocs. When used as coagulant aids, synthetic polyelectrolytes showed little effects on the improvement in organic removal. On the other hand, cationic polyacrylamide was observed to substantially enhance the settleability of flocs as compared to anionic polyacrylamide. The effects of rapid mixing conditions and polymer flocculants on the coagulation performance were discussed. (C) 2009 Elsevier B.V. All rights reserved.

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- SD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Using drift nets to capture early life stages and monitor spawning of the Yangtze River Chinese sturgeon (Acipenser sinensis)

P>A sampling system for capturing sturgeon eggs using a D-shaped bottom anchored drift net was used to capture early life stages (ELS) of Chinese sturgeon, Acipenser sinensis, and monitor annual spawning success at Yichang on the Yangtze River, 1996-2004, before and just after the Three Gorges Dam began operation. Captured were 96 875 ELS (early life stages: eggs, yolk-sac larvae = eleuthero embryos, and larvae); most were eggs and only 2477 were yolk-sac larvae. Most ELS were captured in the main river channel and inside the bend at the Yichang spawning reach. Yolk-sac larvae were captured for a maximum of 3 days after hatching began, indicating quick dispersal downstream. The back-calculated day of egg fertilization over the eight years indicated a maximum spawning window of 23 days (20 October-10 November). Spawning in all years was restricted temporally, occurred mostly at night and during one or two spawning periods, each lasting several days. The brief temporal spawning window may reduce egg predation by opportunistic predators by flooding the river bottom with millions of eggs. During 1996-2002, the percentage of fertilized eggs in an annual 20-egg sample was between 63.5 to 94.1%; however, in 2003 the percentage fertilized was only 23.8%. This sudden decline may be related to the altered environmental conditions at Yichang caused by operation of the Three Gorges Dam. Further studies are needed to monitor spawning and changes in egg fertilization in this threatened population.

Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

The youngest split in sympatric schizothoracine fish (Cyprinidae) is shaped by ecological adaptations in a Tibetan Plateau glacier lake

Although new empirical evidence shows that sympatric speciation has occurred in some species, there are few indisputable model organisms for this process of speciation. The two subspecies (Gymnocypris eckloni eckloni and G. e. scoliostomus) of the schizothoracine Gymnocypris fish species complex from a small glacier lake in the Tibetan Plateau, Lake Sunmcuo, fit several of the key characteristics of the sympatric speciation model. We used combined mitochondrial control region sequences and the cytochrome b gene (1894 bp) to address the phylogenetics and population genetics of 232 specimens of G. e. eckloni and G. e. scoliostomus, as well as all of its closely related sister species. We found that: (i) a total of four old lineages were uncovered in the widespread G. e. eckloni, of which only one was shown to be shared with all G. e. scoliostomus individuals and (ii) the new subspecies (G. e. scoliostomus) evolved in Lake Sunmcuo from the ancestral G. e. eckloni population within approximately 0.057 Ma. These two taxa of the species complex are morphologically distinct, and reproductive isolation is further suggested. Ecological disruptive selection based on morphological traits (e.g. mouth cleft characters) and food utilization may be a mechanism of incipient speciation of two sympatric populations within Lake Sunmcuo. This study provides the first genetic evidence for sympatric speciation in the schizothoracine fish.

The influence of topography and land use on water quality of Xiangxi River in Three Gorges Reservoir region

A self-organizing map (SOM) was used to cluster the water quality data of Xiangxi River in the Three Gorges Reservoir region. The results showed that 81 sampling sites could be divided into several groups representing different land use types. The forest dominated region had low concentrations of most nutrient variables except COD, whereas the agricultural region had high concentrations of NO3N, TN, Alkalinity, and Hardness. The sites downstream of an urban area were high in NH3N, NO2N, PO4P and TP. Redundancy analysis was used to identify the individual effects of topography and land use on river water quality. The results revealed that the watershed factors accounted for 61.7% variations of water quality in the Xiangxi River. Specifically, topographical characteristics explained 26.0% variations of water quality, land use explained 10.2%, and topography and land use together explained 25.5%. More than 50% of the variation in most water quality variables was explained by watershed characteristics. However, water quality variables which are strongly influenced by urban and industrial point source pollution (NH3N, NO2N, PO4P and TP) were not as well correlated with watershed characteristics.