Community Composition of Photosynthetic Picoeukaryotes in a Subtropical Coastal Ecosystem, with Particular Emphasis on Micromonas.

Photosynthetic picoeukaryotes (PPEs) are important constituents in picoplankton communities in many marine ecosystems. However, little is known about their community composition in the subtropical coastal waters of the Northwestern Pacific Ocean. In order to study their taxonomic composition, this study constructed 18S rRNA gene libraries using flow cytometric sorting during the warm season. The results show that, after diatoms, prasinophyte clones are numerically dominant. Within prasinophytes, Micromonas produced the most common sequences, and included clades II, III, IV, and VI. We are establishing the new Micromonas clade VI based on our phylogenetic analysis. Sequences of this clade have previously been retrieved from the South China Sea and Red Sea, indicating a worldwide distribution, but this is the first study to detect clade VI in the coastal waters of Taiwan. The TSA-FISH results indicated that Micromonas clade VI peaked in the summer (~4 × 102  cells/ml), accounting for one-fifth of Micromonas abundance on average. Overall, Micromonas contributed half of Mamiellophyceae abundance, while Mamiellophyceae contributed 40% of PPE abundance. This study demonstrates the importance of Micromonas within the Mamiellophyceae in a subtropical coastal ecosystem.

Strains of the Morphospecies Ploeotia costata (Euglenozoa) Isolated from the Western North Pacific (Taiwan) Reveal Substantial Genetic Differences.

Two phagotrophic euglenid strains (Strains Pac and Tam) were isolated from coastal locations in Taiwan. Ultrastructural characteristics of the strains included five pellicle strips joined at the posterior end. The strips were formed by major grooves with bifurcated edges. At the cell anterior, the feeding structure formed a lip. Underneath the lip was a comb composed of layers of microtubules. Farther back, two supporting rods tapered toward the posterior end, and a number of vanes with attached microtubules were present between the rods. The morphological characteristics agree with Ploeotia costata Strain CCAP 1265/1. However, the 18S rDNA sequences of Strains Pac/Tam lacked a group I intron and possessed three extra insertions of 116, 67, and 53 bp. Phylogenetic analysis indicated low sequence similarity between Strains Pac/Tam and CCAP 1265/1 (92%). The morphospecies P. costata apparently includes a substantial level of DNA sequence divergence, and likely represents multiple molecular species units.

Changes in the Synechococcus Assemblage Composition at the Surface of the East China Sea Due to Flooding of the Changjiang River.

The aim of this study was to elucidate how flooding of the Changjiang River affects the assemblage composition of phycoerythrin-rich (PE-rich) Synechococcus at the surface of the East China Sea (ECS). During non-flooding summers (e.g., 2009), PE-rich Synechococcus usually thrive at the outer edge of the Changjiang River diluted water coverage (CDW; salinity ≤31 PSU). In the summer of 2010, a severe flood occurred in the Changjiang River basin. The plentiful freshwater injection resulted in the expansion of the CDW over half of the ECS and caused PE-rich cells to show a uniform distribution pattern, with decreased abundance compared with the non-flooding summer. The phylogenetic diversity of 16S rRNA gene sequences indicated that the flooding event also shifted the picoplankton community composition from being dominated by Synechococcus, mainly attributed to the clade II lineage, to various orders of heterotrophic bacteria, including Actinobacteria, Flavobacteria, α-Proteobacteria, and γ-Proteobacteria. As an increasing number of studies have proposed that global warming might result in more frequent floods, combining this perspective with the information obtained from our previous [1] and this studies yield a more comprehensive understanding of the relationship between the composition of the marine Synechococcus assemblage and global environmental changes.

Influence of the Changjiang River flood on Synechococcus ecology in the surface waters of the East China Sea.

Synechococcus spp. have been suggested as the primary component of picophytoplankton in the East China Sea (ECS). However, the influences of sudden environmental changes on Synechococcus assemblage composition have not yet been investigated. In the summer of 2010, a disastrous flood occurred in the Changjiang River basin. To improve our understanding of how this flood affected the Synechococcus ecology on the ECS surface, their assemblages and distributions have been described using two-laser flow cytometry and phylogenetic analysis of the phycocyanin operon. During the nonflooding summer of 2009, phycoerythrin-rich (PE-rich) Synechococcus thrived near the outer boundary of the Changjiang River diluted water (CDW) coverage, while phycocyanin-rich (PC-rich) Synechococcus predominated inside the turbid CDW with a transparency of <80%. During the 2010 summer, flooding expanded the CDW coverage area to over half of the ECS. PE-rich cells showed a homogeneous distribution and a decline in abundance, while the spatial pattern of the PC-rich Synechococcus resembled the pattern from 2009. Based on the phycocyanin operon phylogeny, the Synechococcus in the ECS were categorized into five groups, ECS-1 to ECS-4 and ECS-PE, comprising a total of 19 operational taxonomic units. In the summer of 2009, ECS-2 dominated in the coast, and the ECS-3 and ECS-PE clades prevailed in the offshore waters. However, during the summer of 2010, ECS-4 and ECS-PE became the dominant strains. The injection of abundant anthropogenic pollutants and the enhancement of transparency within the CDW expansion area appear to be the factors needed to transiently alter the ecology of Synechococcus after flooding.

Seasonal Patterns of Picocyanobacterial Community Structure in the Kuroshio Current.

The nutrient-scarce, warm, and high-salinity Kuroshio current has a profound impact on both the marine ecology of the northwestern Pacific Ocean and the global climate. This study aims to reveal the seasonal dynamics of picoplankton in the subtropical Kuroshio current. Our results showed that one of the picocyanobacteria, Synechococcus, mainly distributed in the surface water layer regardless of seasonal changes, and the cell abundance ranged from 104 to 105 cells mL-1. In contrast, the maximum concentration of the other picocyanobacteria, Prochlorococcus, was maintained at more than 105 cells mL-1 throughout the year. In the summer and the autumn, Prochlorococcus were mainly concentrated at the water layer near the bottom of the euphotic zone. They were evenly distributed in the euphotic zone in the spring and winter. The stirring effect caused by the monsoon determined their distribution in the water column. In addition, the results of 16S rRNA gene diversity analysis showed that the seasonal changes in the relative abundance of Synechococcus and Prochlorococcus in the surface water of each station accounted for 20 to 40% of the total reads. The clade II of Synechococcus and the High-light II of Prochlorococcus were the dominant strains in the waters all year round. Regarding other picoplankton, Proteobacteria and Actinobacteria occupied 45% and 10% of the total picoplankton in the four seasons. These data should be helpful for elucidating the impacts of global climate changes on marine ecology and biogeochemical cycles in the Western Boundary Currents in the future.

Distribution patterns and phylogeny of marine stramenopiles in the north pacific ocean.

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml(-1), respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.

Deep brain stimulation rectifies the noisy cortex and irresponsive subthalamus to improve parkinsonian locomotor activities.

The success of deep brain stimulation (DBS) therapy indicates that Parkinson's disease is a brain rhythm disorder. However, the manifestations of the erroneous rhythms corrected by DBS remain to be established. We found that augmentation of α rhythms and α coherence between the motor cortex (MC) and the subthalamic nucleus (STN) is characteristically prokinetic and is decreased in parkinsonian rats. In multi-unit recordings, movement is normally associated with increased changes in spatiotemporal activities rather than overall spike rates in MC. In parkinsonian rats, MC shows higher spike rates at rest but less spatiotemporal activity changes upon movement, and STN burst discharges are more prevalent, longer lasting, and less responsive to MC inputs. DBS at STN rectifies the foregoing pathological MC-STN oscillations and consequently locomotor deficits, yet overstimulation may cause behavioral restlessness. These results indicate that delicate electrophysiological considerations at both cortical and subcortical levels should be exercised for optimal DBS therapy.

Nitric oxide as a signaling factor to upregulate the death-specific protein in a marine diatom, Skeletonema costatum, during blockage of electron flow in photosynthesis.

To determine the physiological functions of a novel death-specific protein gene, Skeletonema costatum DSP-1 (ScDSP-1) in a marine diatom, Skeletonema costatum, the mRNA abundance of ScDSP-1 was measured in cultures subjected to light manipulation and treatments with various chemicals. When cells were transferred to a dim light intensity of 15 micromol m(-2) s(-1), ScDSP-1 mRNA levels showed a transient increase of 1 to 17.2 micromol (mol 18S rRNA)(-1) in 60 h. Furthermore, treatments with the photoinhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) resulted in high ScDSP-1 mRNA levels, which reached 943 and 72 micromol (mol 18S rRNA)(-1), respectively. Treatment with the nitric oxide (NO) donor diethylamine nitric oxide also induced ScDSP-1 expression, and this inducible expression was inhibited by the NO scavenger hemoglobin. Additionally, the expression of ScDSP-1 mRNA elicited by DCMU and DBMIB was efficiently reduced when cultures were pretreated with the cell-penetrating NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. In contrast, treatment with another photoinhibitor, paraquat, had no effect on ScDSP-1 expression. Our results indicated that NO is the crucial secondary messenger which signals the expression of ScDSP-1 when electron flow between photosystem II and photosystem I is blocked in S. costatum cells. In addition, the discovery of a similar gene, ScDSP-2, is briefly described.

Parastrombidinopsis minima n. sp. (Ciliophora: Oligotrichia) from the coastal waters of northeastern Taiwan: morphology and small subunit ribosomal DNA sequence.

Parastrombidinopsis minima n. sp. is investigated, using live observations, protargol preparations, and molecular data. In living cells, the ranges of cell length are 85-95 microm, cell width 60-70 microm, and oral diameter 40-50 microm. In protargol-impregnated specimens, cell length ranges between 43 and 71 microm, cell width between 23 and 42 microm, and oral diameter between 13 and 24 microm. The numbers of external oral polykinetids are 12-16 and of somatic kineties are 11-13. There are always two ovoid macronuclei (9-16 x 4-9 microm). Based on the analysis of morphologic data, the new species can be placed in the family Strombidinopsidae, but based on the small subunit rRNA gene sequence data, the Parastrombidinopsis species are more closely associated with strobilidiids and tintinnids.

Predator and prey biodiversity relationship and its consequences on marine ecosystem functioning-interplay between nanoflagellates and bacterioplankton.

The importance of biodiversity effects on ecosystem functioning across trophic levels, especially via predatory-prey interactions, is receiving increased recognition. However, this topic has rarely been explored for marine microbes, even though microbial biodiversity contributes significantly to marine ecosystem function and energy flows. Here we examined diversity and biomass of bacteria (prey) and nanoflagellates (predators), as well as their effects on trophic transfer efficiency in the East China Sea. Specifically, we investigated: (i) predator diversity effects on prey biomass and trophic transfer efficiency (using the biomass ratio of predator/prey as a proxy), (ii) prey diversity effects on predator biomass and trophic transfer efficiency, and (iii) the relationship between predator and prey diversity. We found higher prey diversity enhanced both diversity and biomass of predators, as well as trophic transfer efficiency, which may arise from more balanced diet and/or enhanced niche complementarity owing to higher prey diversity. By contrast, no clear effect was detected for predator diversity on prey biomass and transfer efficiency. Notably, we found prey diversity effects on predator-prey interactions; whereas, we found no significant diversity effect on biomass within the same trophic level. Our findings highlight the importance of considering multi-trophic biodiversity effects on ecosystem functioning in natural ecosystems.

Effects of Asian dust storms on synechococcus populations in the subtropical Kuroshio Current.

Asian dust storms (ADSs) are the major source of dust deposition in the Northwest Pacific Ocean. To gain a better understanding on how ADSs affect the ecology of picophytoplankton in this oligotrophic region, five oceanographic cruises were conducted between March 15 and April 15, 2006 on a segment of the Kuroshio Current near the shelf break of the East China Sea (25.05° N, 123.15° E). During the study period, three ADS events were recorded and increases in nutrient concentrations as well as mixing depths were observed. Most of the ADS events stimulated the growth of Synechococcus, but the abundance of Prochlorococcus either remained unaffected or showed mild declines. A more detailed study was conducted during the ADS event between March 16 and 19. Phylogenetic analysis of 16S ribosomal RNA nucleotide sequences revealed that most of the newly appeared Synechococcus belonged to the clade II lineage. Furthermore, messenger RNA (mRNA) levels of three nutrient deficiency indicators, including idiA (an iron deficiency indicator), ntcA (a nitrogen deficiency indicator), and pstS (a phosphorus deficiency indicator), were analyzed by real-time quantitative reverse-transcription polymerase chain reaction. As this ADS event proceeded, mRNA levels of all these indicators decreased from relatively high to non-detectable values. These results suggest that the contributions of iron, nitrogen, and phosphate by the dust deposition from ADSs promote the growth of Synechococcus in the Kuroshio Current.

The identification of three novel genes involved in the rapid-growth regulation in a marine diatom, Skeletonema costatum.

In order to understand the molecular mechanism of growth regulation in phytoplankton and to develop novel growth-status indicators, a subtraction cDNA library was constructed by using the mRNA extracted from Skeletonema costatum in the rapid-growth stage (RG stage), and three RG-stage-related cDNA fragments, RG#14, RG#25, and RG#42, were obtained. According to the results of sequence analysis, RG#42 belonged to the MCM2-7 protein family, and the other two fragments, RG#14 and RG#25, were novel molecules. Under continuous illumination, these RG-stage-related mRNA expression levels increased from 100- (RG#14 and RG#42) to 1,000-fold (RG#25) with increasing growth rate. Furthermore, under a diel rhythm of light (light-dark = 12:12 h), the daily mean mRNA abundances of RG#14 and RG#25 in the exponential phase also differed from those in the late-stationary phase. However, such differences between these growth phases were not observed in the mRNA levels of RG#42 and PCNA. This study not only provided a new way to investigate the regulatory mechanisms of cell growth but also offered a possibility of employing these gene fragments as indicators to monitor the growth status of phytoplankton in the marine environment.

Zebrafish cdx1b regulates expression of downstream factors of Nodal signaling during early endoderm formation.

We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs.

Cooccurrence of ScDSP gene expression, cell death, and DNA fragmentation in a marine diatom, Skeletonema costatum.

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5' and 3' untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day(-1), the ScDSP mRNA level was at 2 mumol . mole 18S rRNA(-1). In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day(-1), ScDSP mRNA increased dramatically to 668 mumol . mole 18S rRNA(-1), and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.

Identification of a high-affinity phosphate transporter gene in a prasinophyte alga, Tetraselmis chui, and its expression under nutrient limitation.

A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui. The full-length cDNA of TcPHO obtained by 5' and 3' rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids. The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively. In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4. The expression of TcPHO mRNA was dependent on phosphate availability. With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol micro g of total RNA(-1) from day 1 to day 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression. In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol micro g of total RNA(-1)) under low-nitrate conditions. The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T. chui.