Molecular Comparisons of Freshwater and Marine Isolates of the Same Morphospecies of Heterotrophic Flagellates

Heterotrophic flagellates are key components of all ecosystems. Understanding the patterns of biodiversity of these organisms is thus particularly important. Here we analyzed the intraspecific diversity of 10 morphospecies of heterotrophic flagellates comprising representatives of the Apusozoa (2 morphospecies) and Kinetoplastea (8 morphospecies), all belonging to the most common flagellates with worldwide distribution. Most morphospecies showed a mixing of lineages isolated from diverse habitats, indicating that some lineages of these morphospecies had been able to colonize different habitats several times. Furthermore, our results revealed remarkable levels of genetic divergence within most of the morphospecies studied, underlining the difficulty of correctly determining species by means of morphology alone. Many cryptic or pseudocryptic species seem to occur. Our results revealed clear divergence between marine and freshwater lineages of the morphospecies Ancyromonas sigmoides, showing that freshwater lineages have not been able to colonize marine environments and marine lineages have not been able to colonize freshwater environments for a long time.     

异养鞭毛虫原位生长研究进展

近年来,异养鞭毛虫随水体生态内浮游动物小型化进程的加剧,所占比重越来越,已成为当前生态学研究的重要对象,其研究方法也伴随着发生了革命性变化,通过简单介绍异养鞭毛虫的原位研究方法,并就近来研究成果进行了系统综述。     

Composition of Heterotrophic Flagellates in Coastal Waters of Different Trophic Status

Heterotrophic flagellates (HFs) are important members of the aquatic microbial food web. However, information on their spatial patterns in relation to eutrophication is limited. Here, we examined the composition and spatial distributions of HFs (<3 μm) in subtropical coastal waters of different trophic status by re-analyzing two previously published small subunit rDNA pyrosequence datasets using information from the newly launched Protist Ribosomal Reference database (PR²). Whereas the contributions of different major clades composing the Marine Stramenopiles (MASTs), picobiliphytes and Chrysophyceae were found relatively comparable between the stations, contrasting compositions of the Marine Alveolates (MALV) groups I and II were observed. The high and relatively stable contribution of MAST-1, -3 and -7 among the MASTs in both stations suggest their importance as bacterial grazers in coastal waters, irrespective of trophic status. By contrast, the dominance of clades 3, 5 and 14 of MALV II in the eutrophic station implies their importance in regulating the dinoflagellate population at the site. Our study provides insights into the ecological importance of different HF groups in eutrophic coastal ecosystems.     

Occurrence of Marine Birnavirus Through the Year in Coastal Seawater in the Uwa Sea

This study aims to determine the seasonal occurrence of marine birnavirus (MABV) at a coastal site in the Uwa Sea, Japan, in 1997 and 1998. To detect MABV from seawater, a simple method was developed for concentrating MABV by dialysis and ethanol precipitation. The concentrated virus was used for polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and virus isolation. Viral genome was detected through the experimental period. The amount of PCR product varied; it was small in summer, but increased from fall to winter. Viral protein was also detected, and the amount in the January sample was equivalent to approximately 10² TCID₅₀ (50% tissue culture infectious dose) of the virus. However, infectious viruses were not isolated. This suggested that MABV was released from hosts to environmental seawater in winter and possibly degraded after release. Received May 7, 1999; accepted September 16, 1999.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

[This corrects the article on p. 1964 in vol. 58.].     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.     

Artificially Induced Symbiosis Between Marine Flagellates and Vertebrate Tissues in Culture*

SYNOPSIS. Algal symbiotes isolated from marine invertebrate hosts, and grown axenically in culture, have been successfully combined in artificial associations with chick fibroblasts and grunt (Haemulon sp.) fin explants. Growth (measured by cell numbers and chlorophyll a) is stimulated in both symbiotes and host cells. Studies of nitrogen uptake and exchange suggest that these artificial associations are similar to other, natural symbioses involving invertebrate hosts, e.g. the hermatypic corals.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns

The enhanced mineralization of immobilized nitrogen by bacteriophagous protozoa has been thought to favor the nitrification process in soils in which nitrifying bacteria must compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the flagellate Adriamonas peritocrescens on the competition for ammonium between the chemolithotrophic species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi was studied in soil columns, which were continuously percolated with media containing 5 mM ammonium and different amounts of glucose at a dilution rate of 0.007 h^-1 (liquid volumes). A. globiformis won the competition for ammonium. The grazing activities of the flagellates had two prominent effects on the competition between N. europaea and A. globiformis. First, the distribution of ammonium over the profile of the soil columns was more uniform in the presence of flagellates than in their absence. In the absence of flagellates, relatively high amounts of ammonium accumulated in the upper layer (0 to 3 cm), whereas in the underlying layers the ammonium concentrations were low. In the presence of flagellates, however, considerable amounts of ammonium were found in the lower layers, whereas less ammonium accumulated in the upper layer. Second, the potential ammonium-oxidizing activity of N. europaea was stimulated in the presence of flagellates. The numbers of N. europaea at different glucose concentrations in the presence of flagellates were comparable to those in the absence of protozoa. However, in the presence of flagellates, the potential ammonium-oxidizing activities were four to five times greater than those in the absence of protozoa.     

Clustering of Marine Bacteria in Seawater Enrichments

Seawater enrichments of marine bacteria clustered in 20- to 50-(mu)m-wide bands near air-water interfaces. The cells within the band travelled at up to 212 (mu)m s(sup-1) and at an average speed of 163 (mu)m s(sup-1). Mean cell speeds peaked mid-run at 187 (mu)m s(sup-1). At the end of the run, bacteria reversed direction rather than randomly reorienting. The duration of the stops during reversal was estimated at 18 ms, six to seven times shorter than that found in enteric bacteria. Cells hundreds of micrometers from the band travelled at half the speed of the bacteria in the band. The fastest isolate from the seawater enrichment was identified as Shewanella putrefaciens and had an average speed of 100 (mu)m s(sup-1) in culture. Air-water interfaces produced no clustering or speed changes in isolates derived from enrichments. Salinity and pH, however, both influenced speed. The speed and reversal times of the seawater enrichments indicate that the bacteria in them are better adapted for clustering around small point sources of nutrients than are either enteric or cultured marine bacteria.     

海水反硝化细菌富集培养及性能研究

目的：建立一种海水反硝化细菌的富集培养方法,为海水反硝化微生物在实际应用中处理硝酸盐氮做基础工作。方法：选取污水处理厂缺氧池活性污泥,利用人工海水富集培养反硝化细菌。结果：经过25d,可以培养出反硝化速率为4.83mg/（gMLSS&#183;h）、反硝化强度为110mg/（L&#183;h）的海水反硝化细菌培养物。结论：该培养物可利用柠檬酸钠、葡萄糖、甲醇、乙醇、可溶性淀粉作为唯一碳源进行反硝化,其中以柠檬酸钠和乙醇作碳源时硝酸盐氮去除效果最佳,经过12h,去除率即可达到100%。pH在7～8范围内时,改变pH对去除率的影响不大,但当pH〈6时,去除率明显下降。温度在35℃时,去除效果最好,并且随着温度的降低,去除率也降低。     

Abundance and Biomass of Heterotrophic Flagellates, and Factors Controlling Their Abundance and Distribution in Sediments of Botany Bay

The abundance and biomass of heterotrophic flagellates were estimated monthly in sediments of Botany Bay during March 1999-February 2000. The annual abundance and biomass were in the ranges of 0.46-4.70 x 10(5) cells/cm(3) and of 0.30-8.61 micro g C/cm(3), respectively. The majority of heterotrophic flagellates (93-100%) were less than 10 mm in length and few flagellates were larger than 10 mm. Of the total microbial carbon biomass, heterotrophic flagellates made up about 5% (but at times up to 35%). The contribution of heterotrophic flagellates varied from month to month, and among the sites. The abundance of heterotrophic flagellates was negatively correlated with sediment grain size and positively correlated with the abundance of bacteria, algae (autotrophic flagellates and diatoms), and their probable grazers. A best subsets regression analysis showed that bacterial and algal abundance are the most important factors controlling the abundance of heterotrophic flagellates. When the previously reported grazing rates on bacteria were applied, heterotrophic flagellates would consume a maximum of 64% of bacterial standing stock daily in Botany Bay, suggesting that heterotrophic flagellates are important as bacterivores. However, the importance of heterotrophic flagellate grazing probably varies significantly among the sites and from month to month.     

Spatial Bias in the Marine Fossil Record

Inference of past and present global biodiversity requires enough global data to distinguish biological pattern from sampling artifact. Pertinently, many studies have exposed correlated relationships between richness and sampling in the fossil record, and methods to circumvent these biases have been proposed. Yet, these studies often ignore paleobiogeography, which is undeniably a critical component of ancient global diversity. Alarmingly, our global analysis of 481,613 marine fossils spread throughout the Phanerozoic reveals that where localities are and how intensively they have been sampled almost completely determines empirical spatial patterns of richness, suggesting no separation of biological pattern from sampling pattern. To overcome this, we analyze diversity using occurrence records drawn from two discrete paleolatitudinal bands which cover the bulk of the fossil data. After correcting the data for sampling bias, we find that these two bands have similar patterns of richness despite markedly different spatial coverage. Our findings suggest that i) long-term diversity trends result from large-scale tectonic evolution of the planet, ii) short-term diversity trends are region-specific, and iii) paleodiversity studies must constrain their analyses to well-sampled regions to uncover patterns not driven by sampling.     

Diversity of Heterotrophic Nitrogen Fixation Genes in a Marine Cyanobacterial Mat

The diversity of nitrogenase genes in a marine cyanobacterial mat was investigated through amplification of a fragment of nifH, which encodes the Fe protein of the nitrogenase complex. The amplified nifH products were characterized by DNA sequencing and were compared with the sequences of nitrogenase genes from cultivated organisms. Phylogenetic analysis showed that similar organisms clustered together, with the exception that anaerobic bacteria clustered together, even though they represented firmicutes, (delta)-proteobacteria, and (gamma)-proteobacteria. Mat nifH sequences were most closely related to those of the anaerobes, with a few being most closely related to the cluster of (gamma)-proteobacteria containing Klebsiella and Azotobacter species. No cyanobacterial nifH sequences were found from the mat collected in November when Microcoleus chthonoplastes was the dominant cyanobacterium, but sequences closely related to the cyanobacterium Lyngbya lagerheimeii were found during summer, when a Lyngbya strain was dominant. The results indicate that there is a high diversity of heterotrophic nitrogen-fixing organisms in marine cyanobacterial mats.     

The Ecological Roles of Heterotrophic Dinoflagellates in Marine Planktonic Community1

Heterotrophic dinoflagellates are ubiquitous and often abundant protists in marine environments. Recently, several novel predator-prey relationships between heterotrophic dinoflagellates and other planktonic organisms have been discovered and shown to have diverse ecological roles. Heterotrophic dinoflagellates are predators on a wide array of prey items, including phytoplankton, copepod eggs, and early naupliar stages. They are in turn important prey for some metazoa. Some heterotrophic dinoflagellates are predators of and simultaneously prey for other dinoflagellates. These newly discovered predator-prey relationships may influence our conventional view of energy flow and carbon cycling in the marine planktonic community.     

Role of Marine Cyanobacteria in Trace Metal Bioavailability in Seawater

In seawater, several trace metals with biological significance are highly complexed with organic matter. Marine cyanobacteria are an important phytoplanktonic group, with the ability to release trace metal-binding compounds to the seawater medium, which in turn modulates their bioavailability and influences their biogeochemical cycles. Such interactions may allow cyanobacteria to more easily access less available trace metals essential for their metabolic processes, or, conversely, keep the toxic forms of the trace metals from reaching intolerable levels. In this minireview, Cu and Fe interactions with cyanobacteria received special attention, although other trace metals (Co, Pb, Zn, and Cd) are also covered. Recent research has shed light on many aspects of trace metal–cyanobacteria ecology in seawater; nevertheless, the biochemical processes behind this dynamics and the structure of the vast majority of the metal binding compounds remain unclear.     

Ubiquity and Diversity of Heterotrophic Bacterial nasA Genes in Diverse Marine Environments

Nitrate uptake by heterotrophic bacteria plays an important role in marine N cycling. However, few studies have investigated the diversity of environmental nitrate assimilating bacteria (NAB). In this study, the diversity and biogeographical distribution of NAB in several global oceans and particularly in the western Pacific marginal seas were investigated using both cultivation and culture-independent molecular approaches. Phylogenetic analyses based on 16S rRNA and nasA (encoding the large subunit of the assimilatory nitrate reductase) gene sequences indicated that the cultivable NAB in South China Sea belonged to the α-Proteobacteria, γ-Proteobacteria and CFB (Cytophaga-Flavobacteria-Bacteroides) bacterial groups. In all the environmental samples of the present study, α-Proteobacteria, γ-Proteobacteria and Bacteroidetes were found to be the dominant nasA-harboring bacteria. Almost all of the α-Proteobacteria OTUs were classified into three Roseobacter-like groups (I to III). Clone library analysis revealed previously underestimated nasA diversity; e.g. the nasA gene sequences affiliated with β-Proteobacteria, ε-Proteobacteria and Lentisphaerae were observed in the field investigation for the first time, to the best of our knowledge. The geographical and vertical distributions of seawater nasA-harboring bacteria indicated that NAB were highly diverse and ubiquitously distributed in the studied marginal seas and world oceans. Niche adaptation and separation and/or limited dispersal might mediate the NAB composition and community structure in different water bodies. In the shallow-water Kueishantao hydrothermal vent environment, chemolithoautotrophic sulfur-oxidizing bacteria were the primary NAB, indicating a unique nitrate-assimilating community in this extreme environment. In the coastal water of the East China Sea, the relative abundance of Alteromonas and Roseobacter-like nasA gene sequences responded closely to algal blooms, indicating that NAB may be active participants contributing to the bloom dynamics. Our statistical results suggested that salinity, temperature and nitrate may be some of the key environmental factors controlling the composition and dynamics of the marine NAB communities.     

Heterotrophic Activity Throughout a Vertical Profile of Seawater and Sediment in Halifax Harbor, Canada

The relative heterotrophic activity of marine microorganisms was determined at two sites by the heterotrophic uptake technique throughout the water column, the sediment-water interface, and the surface layer of sediment. In the water column, uptake was greatest at the surface and steadily decreased with depth. The percentage of the substrate that was respired also decreased with depth from 69 to 56%. The activity of the sediment-water interface was several orders of magnitude greater than that of the overlying water and twice that of the sediment immediately below. Hand-collected water samples carefully taken as close as 1 cm from the sediment-water interface had the same characteristically low activity as the bottom few meters of water. Microautoradiography with ³H-labeled glucose, glutamic acid, or thymidine revealed a general decrease in the percentage of active cells with depth from 35 to <1%. The number of active cells in the interface and sediment averaged <10% of the total population. The data indicate that the sediment-water interface is the most active region in this system due to an increased number of active cells rather than an increased percentage of active cells or increased per-cell activity.