Homology-Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI-TOF/TOF and Automated De Novo Sequencing.

This study developed a multilayered, gel-based, and underivatized strategy for de novo protein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected to de novo interpretation using DeNovo Explorer software. The de novo sequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots from Alexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future.

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry.

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions.

Nutrient sources and composition of recent algal blooms and eutrophication in the northern Jiulong River, Southeast China.

The natural process of eutrophication is accelerated by human activities worldwide that interrupt nutrient biogeochemical cycles. Three algal bloom events have been monitored in the northern tributary of the Jiulong River since 2009. The inflection points in a robust locally-weighted regression analysis (LOESS) of the relationship between TN and TP concentrations in the river water, and a TN:TP comparison with nutrient source loadings, suggested that both external loading and internal nutrient cycling contributed to these algal blooms. Nutrient release from the sediments may have played an important role in regulating the nutrients in the overlying water column. In particular, excessive nutrient inputs from various sources and ubiquitous river damming caused further accumulation of the nutrient loading. In-situ autochthonous primary production was enhanced in this relatively stable "river" to "lake" water body. Thus, attention must be paid to the effects of river damming and the consequent internal nutrient release.

[Study on the linkage between urban built-up land and water quality in the Jiulong River watershed].

Band grouping indices combined with single band characteristic were used to extract urban built-up land based on satellite image in the Jiulong River Watershed. Landscape ecology method and statistical analysis were employed to explore the relationship between urban built-up land and permanganate index, NH4(+) -N, TP concentrations. There were significantly positive correlations between the proportion of urban built-up land and permanganate index, NH4(+) -N, TP (r = 0.701, 0.695, 0.789). It indicates the proportion of urban built-up land areas in the sub-watershed could be an effective indicator of water quality. The largest patch index (LPI) was positively correlated to permanganate index, NH4(+) -N, TP concentrations in the water (r = 0.555, 0.643, 0.722). The landscape shape index(LSI) was positively correlated to permanganate index and TP concentrations in the water (r = 0.564, 0.553). These means the impacts of urban built-up land on water quality are influenced not only by urban built-up land areas but also by spatial patterns. The seasonally linear correlation results show that water quality deteriorates quickly with urban built-up land during the flood season and dry season, and the water is susceptible to eutrophication in both flood and dry seasons. The water quality in most sub-watersheds are impacted by urban built-up land, while the urban built-up land areas of Longmen stream, Su stream and Xiao stream located in headstreams are intensive, which need to be adjusted and controlled to protect the water quality.

Validation of an agricultural non-point source (AGNPS) pollution model for a catchment in the Jiulong River watershed, China.

This study tested and evaluated the agricultural non-point source (AGNPS) model for the Wuchuan catchment, a typical agricultural area in the Jiulong River watershed, Fujian Province, China. The AGNPS model was calibrated and validated for the study area with observed data on ten storms. The data on eight storms in 2002 were used for calibration while data on two storms were used for validation of the model. Considering the lack of water quality data over a long-term series, a novel method, comparing an internal nested catchment with its surrounding catchment, was used to supplement the less long-term series data. Dual calibration and validation of the AGNPS model was obtained by this comparison. The results indicate that the correlation coefficients were 0.99 and 0.98 for runoff, 0.94 and 0.95 for the peak runoff rate of the large catchment and the small catchment, respectively, and 0.76 for the sediment of the small catchment only. Each pair of correlation coefficients is homogeneous for the same event for the two catchments. With the exception of the sediment yield and particulate phosphorus, the peak runoff rate and other nutrients were well predicted. Sensitivity analysis showed that the Soil Conservation Service curve number and rainfall quantity were the most sensitive parameters, which resulted in high output variations. Erosivity and other parameters had little influence on the hydrological and quality outputs.

Influence of N, P additions on the transfer of nickel from phytoplankton to copepods.

We examined the influence of macronutrient (nitrate and phosphate) additions on Ni uptake by phytoplankton (Prorocentrum donghaiense and Skeletonema costatum) and its subsequent transfer to marine copepods (Calanus sinicus and Labidocera euchaeta). Ni uptake by phytoplankton after 24h of exposure was markedly dependent on nutrient conditions, with a higher nutrient quota facilitating Ni accumulation in the algae. Trophic transfer was quantified by measurements of the Ni assimilation efficiency in C. sinicus and L. euchaeta, feeding on the algae under different nutrient treatments. Ni assimilation efficiency generally increased with an increase of nutrient concentration in the algae. A significant positive-correlation was found between the Ni assimilation efficiencies of the copepods and the %intracellular Ni in the algal cells. However, ambient nutritional conditions had little effect on the physiological turnover rate constant of Ni by copepods. Thus, nutrient enrichment may lead to an increase in Ni uptake and transfer in marine plankton.

Bioenergetic responses in green lipped mussels (Perna viridis) as indicators of pollution stress in Xiamen coastal waters, China.

Samples of green lipped mussels (Perna viridis) were collected from three sites (Huoshao, I'Maluan and Tong'an) around Xiamen coastal waters, where levels of various trace organic pollutants have been studied. Samples were also collected at a "cleaner" reference site near Dongshan Island for comparison. Clearance rate, absorption efficiency and oxygen consumption of the mussels were measured under controlled laboratory condition; organic pollutants in their tissues were also analyzed. Scope for growth (SFG) was employed as a general biomarker to evaluate the stress of pollutants. Specimens from Tong'an site had the lowest SFG values (1.14 J/h/g); while specimens from the I'Maluan site and Huoshao site had SFG values of 5.01 and 6.72 J/h/g, respectively. Specimens from Dongshan (reference site) had a relatively high SFG value of 10.96 J/h/g. There was a significant negative correlation between the SFG of the different populations of green lipped mussels and the concentration of DDT in their tissues.

[Photoreduction of Se (VI) by marine algae-transitional metals-light system].

Seven marine phytoplankton, including five green algae (Tetraselmis levis, Chlorella autotrophica, Dunaliella salina, Nannochloropsis sp. and Tetraselmis subcordiformis), one diatom (Phaeodactylum tricornutum), one red alga (Porphyridium purpureum), and three usual transitional metals (Fe(III), Cu(II), Mn(II)) were used to make up marine phytoplankton-light or transitional metals-light or marine phytoplankton-transitional metals-light system. In such system, Se(VI) could be transformed into Se(IV) by photoreduction. The species transformation of selenium could be photo-induced by redox reaction of transitional metals. The photochemical activity of marine phytoplankton was confirmed for the first time, because marine phytoplankton could adsorb and concentrated of selenium, transitional metals and organic substances (including the exudation of algae, as reducing agent) which redox potentials were changed. The ratios of Se(VI) to Se(IV) were dominated by the species, the concentration of marine phytoplankton and transitional metals, and it could be enhanced through increasing the concentration of marine algae or the combined effect from marine algae and transitional metals. After photoreduction by ternary system, the ratio of Se(VI) to Se(IV) ranges from 1.17 to 2.85, which is close to the actual value in euphotic layer of seawater. The photochemical process that is induced by marine algae and transitional metals dominative the leading effects on the distribution of oxidation states of selenium.

[Modelling pollutant loads and management alternatives in Jiulong River watershed with AnnAGNPS].

The modelling package Annualized Agricultural Nonpoint Source Model (AnnAGNPS) was used to predict pollutant loads, and simulate catchment processes and management practices in Jiulong River watershed, a medium-sized mountainous watershed in southeast of China. Four typical sub-watersheds were primarily chosen to calibrate AnnAGNPS model by data collected from storm events during the period of April to September, 2003. The model was further validated in the two biggest branches of Jiulong River watershed, i.e. West river and North river by the data regarding climate, and land using condition in 2002 - 2003. The simulation results show that annual total nitrogen load was 24.76kg/(hm2 x a) and 10.28kg/(hm2 x a) in the West river and North river, respectively, and annual total phosphorus load was 0.67 kg/(hm2 x a) and 0.40 kg/(hm2 x a) in the West river and North river, respectively. With the support of AnnAGNPS model, several management alternatives were separately simulated in the typical sub-watersheds, West river and North river. In the specific cell with cell-ID of 92 in Tianbao and Xiandu sub-watershed, after reforesting in sloping field, runoff surface, sediment yield, total nitrogen load and total phosphorus load cut down with 21.6%, 25.9%, 96% and 79.2%, respectively. In West river, with the cultivation plant changing from banana into rice, the total nitrogen, dissolved nitrogen, total phosphorus and dissolved phosphorus cut down with 23.83%, 25.44%, 9.08% and 19.84%, respectively. In North river, when removing all the hoggerys, nitrogen and dissolved nitrogen cut down with 63.54% and 76.92% , respectively.

Seasonal study on the Cd, Se, and Zn uptake by natural coastal phytoplankton assemblages.

We conducted a seasonal study of the uptake of Cd, Se(IV), and Zn by natural phytoplankton assemblages collected from two locations in Hong Kong coastal waters. Marine phytoplankton in Port Shelter was dominated by diatoms and cyanobacteria, while phytoplankton in Tolo Harbour was dominated by dinoflagellates. The natural phytoplankton assemblages were spiked with macronutrients (N and P) and metals, and the accumulation of these metals was followed for 3 to 5 d in the laboratory. The two coastal systems were mostly P limited, with N limitation observed only during the summer in Tolo Harbour. With the additions of these macronutrients, phytoplankton responded in different ways in their accumulation of trace metals. Cadmium concentrations (normalized to chlorophyll a [chl a] concentration) increased by 25 to 38% with the simultaneous addition of N and P but were roughly constant when N alone was added. In N-limited systems, the Cd concentration increased by 25% with N addition. Concentrations of Se were reduced competitively (by 40-45%) with P addition. Zinc concentrations were reduced by 23 to 38% when both N and P were added, largely because of a major increase in biomass (biodilution). Both Cd and Zn uptake rates increased with increasing growth rates. The uptake rate of Se was not related to the growth rate because of the confounding influence of P addition. The particle concentration effect was observed only for Zn, in which the increasing chl a concentration biodiluted the Zn concentration in the phytoplankton. Overall, our results suggest that macronutrients may affect trace metal accumulation in natural phytoplankton because of their controls on cell growth, which may have implications for our understanding of trace metal dynamics in coastal ecosystems during phytoplankton blooms as well as for the prediction of trophic transfer in food chains.