mardi 21 avril 2009
par   G. Grégori

Adler NE, Schmitt-Jansen M, Altenburger R (2007) Flow cytometry as a tool to study phytotoxic modes of action. Environmental Toxicology and Chemistry 26 :297-306

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The objective of the present investigation was to provide a diagnostic tool for the analysis of phytotoxic interactions between environmental contaminants and algae by application of flow cytometry. Therefore, an experimental design was developed consisting of synchronized Scenedesmus vacuolatus cell populations at defined cell-cycle stages, short-term exposure against different inhibitors with known molecular targets, and fluorochrome labeling of different metabolic processes. To discriminate cells with compromised metabolic processes from intact and metabolically inactive cells, references for every fluorochrome were defined using control and heat-treated populations. The experimental results showed that fluorescence markers are able to detect disturbance of specific cellular characteristics, such as membrane integrity, chlorophyll synthesis, and degradation. A differentiation of impacts on specific metabolic process caused by the reference inhibitors in concentration-dependent patterns could be seen using flow-cytometric fluorochrome analysis. These findings were compared with effects observed for N-phenyl-2-naphthylamine (PNA), a sediment contaminant of high phytotoxicity but unclear mode of action. Rhodamine 123 and cyano-ditolyl tetrazolium chloride detected significant metabolic changes for relevant exposures against PNA, thus pointing to compromised mitochondrial activity and changes in membrane potential as causes of phytotoxicity.

Allers E, Gomez-Consarnau L, Pinhassi J, Gasol JM, Simek K, Pernthaler J (2007) Response of Alteromonadaceae and Rhodobacteriaceae to glucose and phosphorus manipulation in marine mesocosms. Environmental Microbiology 9 :2417-2429

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Microbial successions were studied in experimental mesocosms of marine water in the presence of additional organic carbon (glucose), phosphorus (P) or both. P addition lead to pronounced blooms of phytoplankton and to significantly enhanced bacterial production. Characteristic succession patterns were observed for two phylogenetic groups of bacteria that both transiently formed > 50% of total cells. An initial bloom of bacteria affiliated to the Alteromonadaceae could not be assigned to any specific treatment and was interpreted as a response to the manipulations during mesocosm set-up. These bacteria rapidly declined with the appearance of heterotrophic nanoflagellates, suggesting a negative effect of selective grazing. The persistence of Alteromonadaceae in the microbial assemblages was significantly favored by the presence of additional glucose. During the second half of the experiment, bacteria affiliated to Rhodobacteriaceae formed a dominant component of the experimental assemblages in treatments with addition of P. The community contribution of Rhodobacteriaceae was significantly correlated with chlorophyll a concentrations only in the P-amended mesocosms (r(2) = 0.58). This was more pronounced in the absence of glucose (r(2) = 0.85). The phylogenetic and morphological diversity among Rhodobacteriaceae was high, and treatment-specific temporal successions of genotypes related to Rhodobacteriaceae were observed. We suggest that the observed succession patterns reflect different niche preferences : Alteromonadaceae rapidly responded to disturbance and profited from allochthonous glucose input, whereas Rhodobacteriaceae benefited from the phytoplankton bloom.

Alonso-Saez L, Aristegui J, Pinhassi J, Gomez-Consarnau L, Gonzalez JM, Vaque D, Agusti S, Gasol JM (2007) Bacterial assemblage structure and carbon metabolism along a productivity gradient in the NE Atlantic Ocean. Aquatic Microbial Ecology 46 :43-53

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Bacterioplankton have the potential to significantly affect the cycling of organic matter in the ocean ; however, little is known about the linkage between bacterial assemblage structure and carbon metabolism. In this study, we investigated whether changes in the phylogenetic composition of bacterioplankton were associated with changes in bacterial carbon processing (bacterial production, respiration and biomass) in the subtropical NE Atlantic Ocean. We found consistent differences in the composition of the bacterial assemblage, as revealed by denaturing gradient gel electrophoresis (DGGE) and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), along a gradient from the NW African upwelling to the oligotrophic North Atlantic Subtropical Gyre. The percent contribution of Bacteroidetes, Roseobacter and Gammaproteobacteria significantly increased towards more productive waters, whereas the SAR11 clade of the Alphaproteobacteria remained relatively constant (average 28% of DAPI-stained cells) throughout the area. Changes in the composition of the bacterial assemblage detected by DGGE were weakly but significantly correlated with changes in carbon processing variables. The abundances of Roseobacter and Gammaproteobacteria were highly correlated with the concentration of particulate organic carbon and chlorophyll a, reflecting the affinity of these groups to nutrient-enriched conditions. The abundance of Roseobacter was also positively correlated with heterotrophic bacterial production, suggesting their active participation in carbon processing.

Alonso-Saez L, Balague V, Sa EL, Sanchez O, Gonzalez JM, Pinhassi J, Massana R, Pernthaler J, Pedros-Alio C, Gasol JM (2007) Seasonality in bacterial diversity in north-west Mediterranean coastal waters : assessment through clone libraries, fingerprinting and FISH. Fems Microbiology Ecology 60 :98-112

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We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4’,6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (> 20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversity.

Andrade L, Gonzalez AM, Rezende CE, Suzuki M, Valentin JL, Paranhos R (2007) Distribution of HNA and LNA bacterial groups in the Southwest Atlantic Ocean. Brazilian Journal of Microbiology 38 :330-336

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Bacterioplankton was studied in a large area of Southwest Atlantic Ocean between 13 and 25 degrees S and 28 and 42 degrees W. Samples were collected in 108 stations at 20 m depth. Bacteria were enumerated by flow cytometry after nucleic acid staining with syto13 and two subgroups were differentiated : low nucleic acid content ( LNA) and high nucleic acid content ( HNA) bacteria. Total bacterial numbers varied from 0.37 to 5.53 10(5) cells mL(-1). HNA cells represented 15 to 70% of the total number while LNA cells represented 30 to 85%. Heterotrophic bacterial production was determined by incorporation of tritiated leucine and ranged from 2.7 to 171.07 ng C L-1 h(-1). No significant correlation was found between abundance and production. Nevertheless with support of multivariate analysis between bacterial abundance, bacterial production, chlorophyll a and other oceanographic data the distribution of the groups in two different oceanic provinces could be explained by nutrient availability. HNA bacteria accounted for the high percentage of cells found in the area north of 19 degrees S, linked to higher temperature waters and riverine nutrients inputs. LNA bacteria were the dominant cells south of this latitude and were correlated to the higher values of nitrate found for the same area.

Assuncao P, Rosales RS, Antunes NT, de la Fe C, Poveda JB (2007) Applications of flow cytometry to mycoplasmology. Front Biosci 12 :664-672


Flow cytometry has become a valuable tool in different fields of microbiology, such as clinical microbiology, aquatic and environmental microbiology, food microbiology, and biotechnology. It combines direct and rapid assays to determine numbers, biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population. This review focuses on the applications of flow cytometry to the field of mycoplasmology. It tries to give a scope of the important breakthroughs which occurred in this field in the last decades, and in the advantages of introducing flow cytometry in research and routine diagnostic procedures of mycoplasmas.

Baltar F, Aristegui J, Gasol JM, Hernandez-Leon S, Herndl GJ (2007) Strong coast-ocean and surface-depth gradients in prokaryotic assemblage structure and activity in a coastal transition zone region. Aquatic Microbial Ecology 50 :63-74

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The distribution of marine Crenarchaeota Group 1, marine Euryarchaeota Group II and some major groups of Bacteria (SAR 11, Roseobacter, Gammaproteobacteria and Bacteroidetes) was investigated in the North Atlantic water column (surface to 2000 m depth) along a transect from the coastal waters of the NW African upwelling to the offshore waters of the Canary Coastal Transition Zone (CTZ). Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was used to describe the prokaryotic assemblages. Bulk picoplankton abundance and leucine incorporation were determined. Pronounced changes in prokaryotic assemblage composition were observed from the coast to the open ocean and at the deep chlorophyll maximum (DCM) with decreasing bulk heterotrophic activity. All bacterial groups decreased in absolute abundances from the coast to the open ocean ; both archaeal groups increased towards the open ocean. Prokaryotic abundance and activity decreased 2 and 3 orders of magnitude, respectively, from the surface to 2000 m. Prokaryotic growth rates were high in the mesopelagic zone (similar to 0.13 d(-)1), compared to other reports from the central North Atlantic. SARI 1 in total picoplankton abundance decreased from 42 % in the DCM to 4 % at 2000 m, while marine Crenarchaeota Group I increased from 1 % in the DCM to 39 % in the oxygen minimum layer. A clear influence of the different intermediate water masses was observed on the bulk heterotrophic picoplankton activity, with lower leucine incorporation rates corresponding to layers where patches of Antarctic Intermediate Water were detected. Coast-ocean and surface-depth gradients in bulk prokaryotic abundance and production and assemblage composition were comparable to changes observed in basin-scale studies, pinpointing the CTZs as regions of strong variability in microbial diversity and metabolism.

Benazzi G, Holmes D, Sun T, Mowlem MC, Morgan H (2007) Discrimination and analysis of phytoplankton using a microfluidic cytometer. Iet Nanobiotechnology 1 :94-101

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Identification and analysis of phytoplankton is important for understanding the environmental parameters that are influenced by the oceans, including pollution and climate change. Phytoplanktons are studied at the single cell level using conventional light-field and fluorescence microscopy, but the technique is labour intensive. Flow cytometry enables rapid and quantitative measurements of single cells and is now used as an analytical tool in phytoplankton analysis. However, it has a number of drawbacks, including high cost and portability. We describe the fabrication of a microfluidic (lab-on-a-chip) device for high-speed analysis of single phytoplankton. The device measures fluorescence (at three wavelength ranges) and the electrical impedance of single particles. The system was tested using a mixture of three, algae (Isochrysis Galbana, Rhodosorus m., Synechococcus sp.) and the results compared with predictions from theory and measurements using a commercial flow cytometer (131) FACSAria). It is shown that the microfluidic flow cytometer is able to distinguish and characterise these different taxa and that impedance spectroscopy enables measurement of phytoplankton biophysical properties.

Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73 :3283-3290


The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

Berney M, Weilenmann HU, Egli T (2007) Adaptation to UVA radiation of E. coli growing in continuous culture. J Photochem Photobiol B 86 :149-159


Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50W/m(2). Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7h(-1), 0.5h(-1) and 0.3h(-1) were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells (D=0.05h(-1)) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3h(-1) only a small fraction (1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.

Bosco D, Rouiller DG, Halban PA (2007) Differential expression of E-cadherin at the surface of rat beta-cells as a marker of functional heterogeneity. J Endocrinol 194 :21-29


The aim of this study was to assess whether the expression of E-cadherin at the surface of rat beta-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under standard conditions, virtually all beta-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two beta-cell sub-populations were sorted : one that was poorly labeled (’ECad-low’) and another that was highly labeled (’ECad-high’). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high beta-cells was higher than that from ECad-low beta-cells. Ca2+-dependent beta-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of beta-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochalasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet beta-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of beta-cell function.

Bouvier T, Del Giorgio PA, Gasol JM (2007) A comparative study of the cytometric characteristics of high and low nucleic-acid bacterioplankton cells from different aquatic ecosystems. Environ Microbiol 9 :2050-2066


Flow cytometry has revealed the existence of two distinct fractions of bacterioplankton cells, characterized by high and low nucleic acid contents (HNA and LNA cells). Although these fractions seem ubiquitous in aquatic systems, little is known concerning the variation in the cytometric parameters used to characterize them. We have performed cytometric analyses of samples from a wide range of aquatic systems to determine the magnitude and variability in the cytometric characteristics of HNA/LNA. We show that neither group is associated to a fixed level of fluorescence and of light scatter. Rather, the relative position of HNA and LNA in the fluorescence versus side scatter cytograms varies greatly, both within and among ecosystems. Although the cytometric parameters of both groups tend to covary, there is often uncoupling between the two, particularly in light scatter. Our results show that, although the basic HNA/LNA configuration is present in most samples, its cytometric expression changes greatly in different ecosystems and along productivity gradients. The patterns in cytometric parameters do not support the simple, dichotomous view of HNA and LNA as active and inactive cells, or the notion of two distinct and independent communities, but rather suggest that there may be cells that are intrinsic to each fraction, as well as others that may exchange between fractions.

Brunet C, Casotti R, Vantrepotte V, Conversano F (2007) Vertical variability and diel dynamics of picophytoplankton in the Strait of Sicily, Mediterranean Sea, in summer. Marine Ecology-Progress Series 346 :15-26

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Phytoplankton pigment diversity and photoacclimation during the natural day-night cycle was investigated at a fixed location in the Strait of Sicily in July 1997 using HPLC pigment analysis on fractionated samples (<3 and >3 mu m) and flow cytometry. Picophytoplankton dominated phytoplankton biomass in terms of chl a with an average value of 57% and was mainly represented by prokaryotes, prymnesiophytes and pelagophytes. Prochlorococcus and picoeukaryotes contributed equally to the picophytoplankton in terms of chl a, but Prochlorococcus were numerically more abundant and were represented by 2 ecotypes, one replacing the other according to depth. Larger phytoplankton were dominated by prymnesiophytes and diatoms. Photoacclimation was evident from changes in pigment content and strongly increased with depth. The deep chlorophyll maximum (DCM), present between 75 and 90 m, showed a diverse and rich phytoplankton community with the 2 size classes almost equally represented. Growth rates of Prochlorococcus and Synechococcus, estimated from cell cycle measurements, were 0.67 and 0.41 d(-1), respectively, at 75 m. Only Prochlorococcus was able to sustain a good growth rate of 0.43 d(-1) at the base of the DCM (90 m) where only 0.5% of incident light was available. Light-shift experiments using onboard incubated natural seawater showed much faster kinetic coefficients for acclimation in picophytoplankton than in larger algae. In general, the data describe the dynamics of picophytoplankton and its light adaptation through the water column and in the DCM, and can be considered representative of stable summer conditions in the Mediterranean Sea.

Bugge DA, Hegaret H, Wikfors GH, Allam B (2007) Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes. Fish & Shellfish Immunology 23 :188-196

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Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorgansisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes ; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites. (c) 2006 Elsevier Ltd. All rights reserved.

Bugge DM, Hegaret H, Wikfors GH, Allam B (2007) Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes. Fish Shellfish Immunol 23 :188-196


Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorganisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes ; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.

Burbage CD, Binder BJ (2007) Relationship between cell cycle and light-limited growth rate in oceanic Prochlorococcus (MIT9312) and Synechococcus (WH8103) (cyanobacteria). Journal of Phycology 43 :266-274

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The relationships between growth rate, cell-cycle parameters, and cell size were examined in two unicellular cyanobacteria representative of open-ocean environments : Prochlorococcus (strain MIT9312) and Synechococcus (strain WH8103). Chromosome replication time, C, was constrained to a fairly narrow range of values (similar to 4-6 h) in both species and did not appear to vary with growth rate. In contrast, the pre- and post-DNA replication periods, B and D, respectively, decreased with increasing growth rate from maxima of similar to 30 and 10-20 h to minima of similar to 4-6 and 2-3 h, respectively. The combined duration of the chromosome replication and postreplication periods (C+D), a quantity often used in the estimation of Prochlorococcus in situ growth rates, varied similar to 2.4-fold over the range of growth rates examined. This finding suggests that assumptions of invariant C+D may adversely influence Prochlorococcus growth rate estimates. In both strains, cell mass was the greatest in slowly growing cells and decreased 2- to 3-fold over the range of growth rates examined here. Estimated cell mass at the start of replication appeared to decrease with increasing growth rate, indicating that the initiation of chromosome replication in Prochlorococcus and Synechococcus is not a simple function of cell biomass, as suggested previously. Taken together, our results reflect a notable degree of similarity between oceanic Synechococcus and Prochlorococcus strains with respect to their growth-rate-specific cell-cycle characteristics.

Caro A, Gros O, Got P, De Wit R, Troussellier M (2007) Characterization of the population of the sulfur-oxidizing symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses. Appl Environ Microbiol 73 :2101-2109


We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 mum in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.

Chen HY, Chu X, Yan CL, Chen XH, Sun M, Wang YJ, Wang CB, Yu WG (2007) Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B. Acta Pharmacol Sin 28 :1665-1670


AIM : Polypeptide from Chlamys farreri (PCF, molecular mass is 879) is a new marine polypeptide compound isolated from Chlamys farreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B (UVB)-induced apoptosis in murine thymocytes. METHODS : The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. RESULTS : It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. CONCLUSION : Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.

Chiang OE, Quinones RA (2007) Relationship between viral and prokaryotic abundance on the Bajo O’Higgins 1 seamount (Humboldt Current System off Chile). Scientia Marina 71 :37-46

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There is little known about the ecology of microbial communities living in the water column over seamounts. Here, for the first time, the spatial distribution and abundance of virus-like particles (VLP) are described over a seamount. The association between VLP distribution, prokaryotic abundance, and environmental variables is also analyzed. Sampling was conducted in December 2004 on the Bajo O’Higgins I seamount (32 degrees 54’S, 73 degrees 53’W) located in the Humboldt Current System off Chile. A oxygen minimum layer (OMZ) was clearly present between 130 and 280 m in the water column over the seamount. Water samples were taken with Niskin bottles at 10 oceanographic stations over the seamount at depths of 5, 20, 50, 75, 100, and 150 m and at the benthic boundary layer (BBL ; 5-12 m over the sediments). Temperature, salinity, oxygen, chlorophyll a, and phaeopigments were measured at each station. Viral and prokaryotic abundances were determined with fluorochrome SYBR Green I. Viral abundance ranged from 1.53 x 10(9) VLP L-1 - 16.48 x 10(9) VLP L-1, whereas prokaryotic abundance ranged from 1.78 x 10(8) cell L-1 - 14.91 x 10(8) cell L-1, The virus-like particle/prokaryote ratio varied widely among the analyzed layers (i.e. surface, OMZ, and BBL), probably due to the presence of different prokaryotic and viral assemblages in each layer. Our results indicate that the environmental conditions, mainly the concentration of dissolved oxygen in the water column over Bajo O’Higgins I seamount, shape the association between viral and prokaryotic abundance.

Choi DH, Jang HN, Ha DM, Kim JW, Oh CH, Choi SH (2007) Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD. J Biochem Mol Biol 40 :459-466


The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the 85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

Colombet J, Robin A, Lavie L, Bettarel Y, Cauchie HM, Sime-Ngando T (2007) Virioplankton ’pegylation’ : Use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems. Journal of Microbiological Methods 71 :212-219

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We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses. (c) 2007 Elsevier B.V. All rights reserved.

Culverhouse P (2007) Human and machine factors in algae monitoring performance. Ecological Informatics 2 :361-366

Du M, Chen J, Zhang X, Li A, Li Y (2007) Characterization and resuscitation of viable but nonculturable Vibrio alginolyticus VIB283. Arch Microbiol 188 :283-288


The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4 degrees C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10(10) to 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.

Du M, Chen JX, Zhang XH, Li AJ, Li Y (2007) Characterization and resuscitation of viable but nonculturable Vibrio alginolyticus VIB283. Archives of Microbiology 188 :283-288

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The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4 degrees C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10(10) to 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.

Dubelaar G, Casotti R, Tarran GA, Biegala IC (2007) Phytoplankton and their analysis by flow cytometry. In : Doleel J, Greilhuber J, Suda J (eds) FLow Cytometry with Plant Cells : Analysis of Genes, Chromosomes and Genomes. WILEY-VCH Verlag GmbH & Co. KgaA, Weinheim, p 287-322

Duhamel S, Moutin T, Van Wambeke F, Van Mooy B, Rimmelin P, Raimbault P, Claustre H (2007) Growth and specific P-uptake rates of bacterial and phytoplanktonic communities in the Southeast Pacific (BIOSOPE cruise). Biogeosciences 4 :941-956

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Predicting heterotrophic bacteria and phytoplankton specific growth rates (mu) is of great scientific interest. Many methods have been developed in order to assess bacterial or phytoplankton mu. One widely used method is to estimate mu from data obtained on biomass or cell abundance and rates of biomass or cell production. According to Kirchman (2002), the most appropriate approach for estimating mu is simply to divide the production rate by the biomass or cell abundance estimate. Most methods using this approach to estimate mu are based on carbon (C) incorporation rates and C biomass measurements. Nevertheless it is also possible to estimate mu using phosphate (P) data. We showed that particulate phosphate (PartP) can be used to estimate biomass and that the P uptake rate to PartP ratio can be employed to assess mu. Contrary to other methods using C, this estimator does not need conversion factors and provides an evaluation of mu for both autotrophic and heterotrophic organisms. We report values of P-based mu in three size fractions (0.2-0.6 ; 0.6-2 and > 2 mu m) along a Southeast Pacific transect, over a wide range of P-replete trophic status. P-based mu values were higher in the 0.6-2 mu m fraction than in the > 2 mu m fraction, suggesting that picoplankton-sized cells grew faster than the larger cells, whatever the trophic regime encountered. Picoplankton-sized cells grew significantly faster in the deep chlorophyll maximum layer than in the upper part of the photic zone in the oligotrophic gyre area, suggesting that picoplankton might outcompete > 2 mu m cells in this particular high-nutrient, low-light environment. P-based mu attributed to free-living bacteria (0.2-0.6 mu m) and picoplankton (0.6-2 mu m) size-fractions were relatively low (0.11+/-0.07 d(-1) and 0.14+/-0.04 d(-1), respectively) in the Southeast Pacific gyre, suggesting that the microbial community turns over very slowly.

Eek KM, Sessions AL, Lies DP (2007) Carbon-isotopic analysis of microbial cells sorted by flow cytometry. Geobiology 5 :85-95

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One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high-precision measurement of C-13 in specific populations of microbial cells that are purified by fluorescence-activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their C-13 content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home-built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of delta C-13 in whole cells with precision better than 0.2%. for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 104 eukaryotic or 107 bacterial cells per sample. Coupled with 13 C-labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM-IRMS also permits useful studies of natural abundance variations in 13C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.

Eller G, Toebe K, Medlin LK (2007) Hierarchical probes at various taxonomic levels in the Haptophyta and a new division level probe for the Heterokonta. Journal of Plankton Research 29 :629-640

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We present a set of specific ribosomal RNA probes for the Haptophyla and a new probe for the Heterokonta, which, in combination with previously described probes, will allow for the rapid stepwise characterization of phytoplankton samples for marine plankton members of these algal divisions by fluorescence in situ hybridization. Probes are described for the Haptophya Classes Plymnesiophyceae and Pavlovaphyceae, for the Order Cocccolithales, for clades known only from clone library sequence data and for the genus Prymnesium. The division level probe for the Heterokonta presented here mismatches only 4.5% of all heterokonts, where the complete 18S rDNA is known. It does not target any organism outside the heterokonts, expect for two environmental clones, which could be chimeras. The heterokonts not targeted by this probe fall mostly in the non-pigmented clades at the base of the heterokont tree. When this probe is coupled with other published class level probes, the blodiversio of the heterokont classes can be addressed by fluorescence in situ hybridizations.

Felip M, Andreatta S, Sommaruga R, Straskrabova V, Catalan J (2007) Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples : Comparison with microscopy data. Applied and Environmental Microbiology 73 :4508-4514

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The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4’,6’-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4’,6’-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R-2 = 0. 545, P < 0.001, n = 80) and the largest (R-2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that How cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.

Fernandez C, Thyssen M, Denis M (2007) Microbial community structure along 18°W (39°N–44.5°N) in the NE Atlantic in late summer 2001 (POMME programme) Journal of Marine Systems 71 :46-62

Franklin NM, Rogers NJ, Apte SC, Batley GE, Gadd GE, Casey PS (2007) Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata) : The importance of particle solubility. Environmental Science & Technology 41 :8484-8490

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Metal oxide nanoparticles are finding increasing application in various commercial products, leading to concerns for their environmental fate and potential toxicity. It is generally assumed that nanoparticles will persist as small particles in aquatic systems and that their bioavailability could be significantly greater than that of larger particles. The current study using nanoparticulate ZnO (ca. 30 nm) has shown that this is not always so. Particle characterization using transmission electron microscopy and dynamic light scattering techniques showed that particle aggregation is significant in a freshwater system, resulting in flocs ranging from several hundred nanometers to several microns. Chemical investigations using equilibrium dialysis demonstrated rapid dissolution of ZnO nanoparticles in a freshwater medium (pH 7.6), with a saturation solubility in the milligram per liter range, similar to that of bulk ZnO. Toxicity experiments using the freshwater alga Pseudokirchneriella subcapitata revealed comparable toxicity for nanoparticulate ZnO, bulk ZnO, and ZnCl2, with a 72-h IC50 value near 60 mu g Zn/L, attributable solely to dissolved zinc. Care therefore needs to be taken in toxicity testing in ascribing toxicity to nanoparticles per se when the effects may be related, at least in part, to simple solubility.

Fujimoto T, Sakao S, Yamaha E, Arai K (2007) Evaluation of different doses of UV irradiation to loach eggs for genetic inactivation of the maternal genome. J Exp Zool Part A Ecol Genet Physiol 307 :449-462


Genetic inactivation of the egg nucleus is an indispensable step in the production of androgenetic embryos in teleosts. However, few experimental studies have focused on determining the most effective means of achieving complete inactivation of the maternal genome. Here, we sought to identify the optimum conditions of ultraviolet (UV) irradiation for complete inactivation of the loach egg nucleus. Unfertilized eggs were UV irradiated from above with a dose in the range 0-200 mJ/cm2. Successful inactivation of the maternal genome was evaluated by the exclusive expression of a paternally inherited color phenotype. The presence or absence of putative maternal chromosome fragments was screened by flow cytometry of DNA content and by cytogenetic analysis. The majority of the larvae derived from irradiated eggs had an abnormal appearance. Haploid individuals were detected by measurement of DNA content flow cytometry and by chromosome counting in the groups that received more than 75 mJ/cm2 groups. Although the coefficient of variation of DNA content was apparently reduced in the 125-200 mJ/cm2 groups, chromosome fragments were still detected in all the groups from irradiated eggs. Inactivation of the egg nucleus was also histologically elucidated by the presence or absence of residual nuclear material in anuclear embryos that developed from UV-irradiated eggs fertilized with UV-irradiated sperm. Embryos that were completely or near-completely anuclear were found in the 150 and 200 mJ/cm2 groups. We conclude that the optimum UV dose for complete genetic inactivation of the egg nucleus is more than 150 mJ/cm2.

Gasol JM, Aristegui J (2007) Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity. Aquatic Microbial Ecology 46 :71-83

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To understand the opposing views on the utility of the CTC method, we examined bacterial abundance over incubations with the fluorogenic tetrazolium dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and inspected the flow cytometric signatures of bacteria dually labeled with a DNA stain (Syto13) and CTC. Incubation of a marine plankton sample with CTC produced positive cells up to a stable value, reached in < 1 h, while the red fluorescence of the granules increased for at least an extra hour. Incubation also produced a decrease in cell abundance of 22% after 30 min in the presence of 5 mM CTC. The decrease was a function of CTC concentration and incubation time, and particularly affected bacteria with high nucleic acid content. Flow cytometric inspection of a double-stained (CTC and Syto13) sample showed that after 15 min of incubation, particles appeared having both red (CTC+) and green (DNA+) staining. Afterwards, other particles appeared that maintained the same light scatter properties and the red fluorescence, but that lost all green fluorescence. While the number of particles with double staining stabilized after about 1 h, particles with red but without DNA staining increased for at least 100 min. Simultaneously, the classic determination of CTC+ cells (observing only the red signal of the particles) increased as reported elsewhere. We interpreted these patterns as evidence of CTF (the formazan derivative of CTC) particles growing in the bacterial cells until they are so large that they break up the cells, after which they remain present as CTF granules with no associated cellular material. Microscopic or flow cytometric enumeration of red particles might still be a good indication of the percentage of bacterial cells having taken up and reduced the activity probe, but flow cytometric cell sorting of red particles based only on scatter and red fluorescence signals will select CTF particles without associated cellular material. Our results help reconcile the ecologically sound results and the CTC toxicity evidence currently reported in the literature, and lead to a warning against interpretations of cell sorting of CTC+ particles for phylogenetic or activity studies based only on red or orange fluorescence.

Gonzalez-Quiros R, Munuera I, Folkvord A (2007) Cell cycle analysis of brain cells as a growth index in larval cod at different feeding conditions and temperatures. Scientia Marina 71 :485-497

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The percentage of cells dividing in a specific tissue of individual larvae can be estimated by analyzing DNA per cell by flow cytometry. An experimental test was carried out with cod (Gadus morhua) larvae, with brain as the target tissue, to validate this technique as an appropriate growth index for larval fish. Standard length (SL), myotome height, and %S-phase (% of cells in the S-phase of the cell-division cycle) variability were analyzed, with temperature (6 and 10 degrees C, food level (high- and no-food) and larval developmental stage (first feeding, pre-metamorphosis and post-metamorphosis) as independent factors. Cod larvae grew faster (in SL) and presented a higher %S-phase under high-food conditions. Larval SL increased with temperature in rearing and experimental ranks. However, there was a significant interaction between temperature and food in the %S-phase. There were no significant differences in the %S-phase between 6 and 10 degrees C at high-food levels. We suggest that this result is a consequence of temperature-dependency of the duration of the cell cycle. In the absence of food, larvae at 10 degrees C had a lower %S-phase than larvae at 6 degrees C, which may be related to increased metabolic costs with increasing temperature. Considering the effect of temperature, the mean % S-phase explained 74% of the variability in the estimated standard growth rate.

Gotz D, Paytubi S, Munro S, Lundgren M, Bernander R, White MF (2007) Responses of hyperthermophilic crenarchaea to UV irradiation. Genome Biol 8 :R220


BACKGROUND : DNA damage leads to cellular responses that include the increased expression of DNA repair genes, repression of DNA replication and alterations in cellular metabolism. Archaeal information processing pathways resemble those in eukaryotes, but archaeal damage response pathways remain poorly understood. RESULTS : We analyzed the transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius. Sulfolobus species encounter high levels of DNA damage in nature, as they inhabit high temperature, aerobic environments and are exposed to sunlight. No increase in expression of DNA repair genes following UV irradiation was observed. There was, however, a clear transcriptional response, including repression of DNA replication and chromatin proteins. Differential effects on the expression of the three transcription factor B (tfb) genes hint at a mechanism for the modulation of transcriptional patterns in response to DNA damage. TFB3, which is strongly induced following UV irradiation, competes with TFB1 for binding to RNA polymerase in vitro, and may act as a repressor of transcription or an alternative transcription factor for certain promoters. CONCLUSION : A clear response to DNA damage was observed, with down-regulation of the DNA replication machinery, changes in transcriptional regulatory proteins, and up-regulation of the biosynthetic enzymes for beta-carotene, which has UV protective properties, and proteins that detoxify reactive oxygen species. However, unlike eukaryotes and bacteria, there was no induction of DNA repair proteins in response to DNA damage, probably because these are expressed constitutively to deal with increased damage arising due to high growth temperatures.

Grob C, Ulloa O, Li WKW, Alarcon G, Fukasawa M, Watanabe S (2007) Picoplankton abundance and biomass across the eastern South Pacific Ocean along latitude 32.5 degrees S. Marine Ecology-Progress Series 332 :53-62

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The distribution of picoplankton (< 2 to 3 pm in diameter) was determined on a transect across the eastern South Pacific Ocean from south of Tahiti to the coast of Chile along 32.5 degrees S latitude during the early austral spring. The abundance of Synechococcus, picophytoeukaryotes and bacterioplankton increased from oligo- to eutrophic conditions, while that of Prochlorococcus decreased according to nutrient availability and hydrographic characteristics. Bacterioplankton dominated across the transect (> 75% total picoplanktonic abundance). As expected, Prochlorococcus was the most numerically abundant phytoplankter under very oligotrophic (chlorophyll a concentration <=> 0.1 and <= 1 mg m(-3)) conditions. However, in contrast to other subtropical regions, picophytoeukaryotes appear to dominate the < 2 mu m autotrophic carbon biomass in this region of the South Pacific Ocean at this time of the year. In the upper 200 m of the water column, the integrated carbon biomass of Prochlorococcus, Synechococcus, picophytoeukaryotes and bacterioplankton were in the ratios of 9:1:14:11 and 3:1:8:6 under oligo- and mesotrophic conditions, respectively. Thus, picophytoeukaryotes were 1.4- to 2-fold higher in biomass than both cyanobacteria combined, and. slightly more important (1.2- to 1.3-fold) than bacterioplankton. Picophytoeukaryotes could therefore play a dominant ecological and biogeochemical role in subtropical gyres, which extend over a vast area of the world’s oceans.

Gruden C, McCulloch R, Towey T, Wolfe J, AdriaenS P (2007) Hydrogen-based activity enhancement in sediment cultures and intact sediments. Environmental Engineering Science 24 :696-706

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The potential for hydrogen gas to stimulate microbial respiratory activity in sediments was investigated. Cell elutions from Passaic River (NJ), San Diego Bay (CA), and Marine Harbor sediments were amended with hydrogen gas to evaluate its impact on microbial activity measured by intracellular reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). The transferability of this approach to sediment slurries and static sediment columns was evaluated based on microbial activity enhancement in Marine Harbor sediments. Results indicate that microbial activity can be increased by a factor of 2-3 at a threshold hydrogen concentration range (0.5 to 1.5 mu M). Terminal restriction fragment (T-RF) length polymorphism analysis indicated that the community response to hydrogen resulted in the emergence of previously recessive populations. The causal relationship between hydrogen amendment and an increase in CTC-active cells was most likely due to community structure shifts, as evidenced by the emergence of new T-RFs (19% of total) at hydrogen concentrations above 1.5 mu M. No RF was dominant within this emergent group, and no chlororespirers were detected within this group, the latter probably due to the lack of enrichment of halogenated compounds. Nevertheless, the transferability of the observed relationship between hydrogen gas amendment and microbial activity to complex sediment samples suggests a promising remedial strategy for in place contaminated estuarine sediments.

Guo N, Zhang X, Lu Y, Song X (2007) Analysis on the factors affecting start-up intensity in the upstream sequence of phycocyanin beta subunit gene from Arthrospira platensis by site-directed mutagenesis. Biotechnol Lett 29 :459-464


Six promoters in the 419 bp upstream sequence of the phycocyanin beta subunit gene of Arthrospira platensis FACHB341 have been previously cloned. Site-directed mutagenesis has now been used to introduce mutations in the -10 and -35 boxes of promoter 3, -10 box of promoter 4, and -35 box of promoter 6. The expression level of green fluorescent protein gene was measured by flow cytometry. Results showed that the effects of site-directed mutagenesis in different promoters were dissimilar : some increased and some declined.

Hammes F, Meylan S, Salhi E, Koster O, Egli T, von Gunten U (2007) Formation of assimilable organic carbon (AOC) and specific natural organic matter (NOM) fractions during ozonation of phytoplankton. Water Res 41 :1447-1454


Ozonation of natural surface water increases the concentration of oxygen-containing low molecular weight compounds. Many of these compounds support microbiological growth and as such are termed assimilable organic carbon (AOC). Phytoplankton can contribute substantially to the organic carbon load when surface water is used as source for drinking water treatment. We have investigated dissolved organic carbon (DOC) formation from the ozonation of a pure culture of Scenedesmus vacuolatus under defined laboratory conditions, using a combination of DOC fractionation, analysis of selected organic acids, aldehydes and ketones, and an AOC bioassay. Ozonation of algae caused a substantial increase in the concentration of DOC and AOC, notably nearly instantaneously upon exposure to ozone. As a result of ozone exposure the algal cells shrunk, without disintegrating entirely, suggesting that DOC from the cell cytoplasm leaked through compromised cell membranes. We have further illustrated that the specific composition of newly formed AOC (as concentration of organic acids, aldehydes and ketones) in ozonated lake water differed in the presence and absence of additional algal biomass. It is therefore conceivable that strategies for the removal of phytoplankton before pre-ozonation should be considered during the design of drinking water treatment installations, particularly when surface water is used.

Harford AJ, O’Halloran K, Wrightt PFA (2007) Effect of in vitro and in vivo organotin exposures on the immune functions of murray cod (Maccullochella peelii peelii). Environmental Toxicology and Chemistry 26 :1649-1656

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Murray cod (Maccullochella peelii peelii) is an iconic native Australian freshwater fish and an ideal species for ecotoxicological testing of environmental pollutants. The species is indigenous to the Murray-Darling basin, which is the largest river system in Australia but also the ultimate sink for many environmental pollutants. The organotins tributyltin (TBT) and dibutyltin (DBT) are common pollutants of both freshwater and marine environments and are also known for their immunotoxicity in both mammals and aquatic organisms. In this study, TBT and DBT were used as exemplar immunotoxins to assess the efficiency of immune function assays (i.e., mitogen-stimulated lymphoproliferation, phagocytosis in head kidney tissue, and serum lysozyme activity) and to compare the sensitivity of Murray cod to other fish species. The organotins were lethal to Murray cod at concentrations previously reported as sublethal in rainbow trout (i.e., intraperitoneal [i.p.] lethal dose to 75% of the Murray cod [LD75] = 2.5 mg/kg DBT and i.p. lethal dose to 100% of the Murray cod [LD100] = 12.5 mg/kg TBT and DBT). In vivo TBT exposure at 0.1 and 0.5 mg/kg stimulated the phagocytic function of Murray cod (F = 6.89, df = 18, p = 0.004), while the highest concentration of 2.5 mg/kg TBT decreased lymphocyte numbers (F = 7.92, df = 18, p = 0.02) and mitogenesis (F = 3.66, df = 18, p = 0.035). Dibutyltin was the more potent immunosuppressant in Murray cod, causing significant reductions in phagocytic activity (F = 5.34, df = 16, p = 0.013) and lymphocyte numbers (F = 10.63, df = 16, p = 0.001).

Hibi K, Mitsubayashi K, Fukuda H, Ushio H, Hayashi T, Ren H, Endo H (2007) Rapid direct determination using combined separation by prepared immunomagnetic and flow cytometry of Flavobacterium psychrophilum. Biosens Bioelectron 22 :1916-1919


Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD), was originally isolated from coho salmon Oncorhychus kisutch in the USA. Bacterial cold-water disease has since been spreading throughout Japan and has caused serious damage to populations of ayu Plecoglossus altivel in many farms and rivers. The rapid method of detecting for F. psuchrophilum is requested, however, traditional methods are laborious because of complicated assay procedures. In this study, a rapid method of detecting F. psychrophilum was developed using a modified method of flow cytometry (FCM) analysis and immunomagnetic separation (IMS). Magnetic iron, in small particles, was prepared by the reaction of a mixture of ferric and ferrous ions under alkaline conditions. The particles were coated with antiserum against F. psychrophilum by dextran. Polyclonal antibodies (anti-F. psychrophilum) conjugated with fluorescein isothiocyanate (FITC) were reacted with F. psychrophilum, and then prepared immunomagnetic were applied using IMS, followed by FCM determination. A good correlation was observed between the cell numbers determined by the FCM method and the traditional method in the range of 10(2)-10(8) cells ml(-1). The FCM analysis could count cells within 1min, and the total analysis time, including sample preparation, was less than 2 h.

Hildebrand M, Frigeri LG, Davis AK (2007) Synchronized growth of Thalassiosira pseudonana (Bacillariophyceae) provides novel insights into cell-wall synthesis processes in relation to the cell cycle. Journal of Phycology 43 :730-740

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Cell-cycle effects in phytoplankton have both general and specific influences over a variety of cellular processes. Understanding these effects requires that the majority of cells in a culture are progressing through the same cell-cycle stage, which requires synchronous growth. We report the development of a silicon starvation-recovery synchrony for the first diatom with a sequenced genome, Thalassiosira pseudonana Hasle et Heimdale, which provides several novel insights into the process of cell-wall formation. After 24 Ir of silicate starvation, flow cytometry measurements indicated that 80% of the cells were arrested in the early G1 phase of the cell cycle and then upon silicate replenishment progressed synchronously through the cycle. An early GI-arrest point was not previously documented in diatoms. After silicate replenishment, girdle-band synthesis was confined to a particular period in G1, and cells did not lengthen in accordance with each girdle band added, which has implications related to cell growth and separation processes in diatoms. Measurements of silicic acid uptake, intracellular pools, and silica incorporation into the cell wall, coupled with fluorescence visualization of newly synthesized cell-wall structures, provide the first direct measurements of silica amounts in individual girdle bands and valves in a diatom. Fluorescence imaging indicated why valves in T. pseudonana do not have to reduce in size with each generation and enabled visualization of intermediates in structure formation. The development of a synchrony procedure for T. pseudonana enables correlation of cellular events with the cell cycle, which should facilitate the use of genomic information.

Hortnagl PH, Sommaruga R (2007) Photo-oxidative stress in symbiotic and aposymbiotic strains of the ciliate Paramecium bursaria. Photochem Photobiol Sci 6 :842-847


We tested the hypothesis that photo-oxidative stress is greater in symbiotic representatives of the freshwater ciliate Paramecium bursaria than in aposymbiotic (i.e., without Chlorella) ones. The level of oxidative stress was determined by assessing reactive oxygen species (ROS) with two fluorescent probes (hydroethidine and dihydrorhodamine123) by flow cytometry in exponential and stationary growth phases of both strains. Photo-oxidative stress was assessed in the laboratory after exposure of the ciliates to photosynthetically active radiation (PAR : 400-700 nm) and PAR+ultraviolet radiation (UVR : 280-400 nm). Additionally, both strains were screened for their antioxidant defenses by measuring the activity of the enzymes catalase, superoxide dismutase (SOD), and glutathione reductase. The results showed that aposymbiotic ciliates had higher levels of PAR-induced oxidative stress than symbiotic ones. Significant differences in PAR-induced oxidative stress were also found in both strains when comparing exponential and stationary growth phases with generally higher values in the former. After exposure to UVR, aposymbiotic ciliates in the stationary phase had the highest levels of ROS despite an increase in SOD activity. By contrast, exposure to UVR decreased catalase activity in both strains. Overall, our results suggest that in this ciliate symbiosis, the presence of symbionts minimizes photo-oxidative stress. This work represents the first assessment of photo-oxidative stress in an algal-ciliate mutualistic symbiosis.

Ivanikova NV, Popels LC, McKay RM, Bullerjahn GS (2007) Lake Superior supports novel clusters of cyanobacterial picoplankton. Appl Environ Microbiol 73 :4055-4065


Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world’s lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the "picocyanobacterial clade" consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10(5) cells ml-1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.

Ivanikova NV, Popels LC, McKay RML, Bullerjahn GS (2007) Lake superior supports novel clusters of cyanobacterial picoplankton. Applied and Environmental Microbiology 73 :4055-4065

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Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world’s lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcRA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the "picocyanobacterial clade" consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and 11, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10(5) cells ml(-1) and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.

Johansson S, Wennergren G, Aberg N, Rudin A (2007) Clara cell 16-kd protein downregulates T-H(2) differentiation of human naive neonatal T cells. Journal of Allergy and Clinical Immunology 120 :308-314

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Background : Levels of the Clara cell 16-kd protein (CC16) are lower in plasma and bronchoalveolar lavage fluid from adults with asthma relative to those seen in healthy control subjects, and CC16 inhibits the T(H)2 cytokine production from marine T cells.
Objective : We sought to determine the plasma levels of CC16 in infants and to investigate whether CC16 might inhibit the T(H)2 cytokine production from neonatal T cells.
Methods : Cord blood and blood samples at 4, 18, and 36 months of age were taken from 64 children prospectively, and CC16 levels were analyzed in plasma. Cord monocyte-derived dendritic cells (DCs) were pulsed with birch allergen extract alone or together with CC16 or prostaglandin D-2 receptor inhibitors, after which autologous naive CD4(+) T cells were added to the DCs. The production of IL-5, IL-13, and IFN-gamma was measured by means of ELISA and flow cytometry.
Results : The plasma levels of CC16 in children peaked at 4 months. CC16 did not directly affect the cytokine production from human T(H)2 cells. However, CC16 inhibited birch pollen extract-stimulated T(H)2 differentiation of naive T cells through the DC. Inhibition of the prostaglandin D-2 receptors did not consistently result in suppressed T(H)2 differentiation.
Conclusion : The production of CC16 seems to peak early in life, and CC16 has an inhibitory effect on T(H)2 cell differentiation from human infants by affecting DCs.
Clinical implications : CC16 is an immunoregulatory protein, and its inhibitory effect on T(H)2 cell differentiation might be of importance in the pathogenesis of allergy in infants.

Johnson HL, Stauber JL, Adams MS, Jolley DF (2007) Copper and zinc tolerance of two tropical microalgae after copper acclimation. Environmental Toxicology 22 :234-244

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Current toxicity tests with microalgae are often criticized as being overly sensitive to metals because algae are cultured in metal-deficient media. If such bioassays overestimate copper toxicity in surface waters, the relevance of water quality guidelines derived from these tests is questionable. In this study, the effect of acclimation to copper at environmentally relevant concentrations, on the sensitivity of the marine diatom Nitzschia closterium and the freshwater green alga Chlorella sp. to copper and zinc was examined. N. closterium was acclimated in culture medium containing 5 or 25 mu g Cu L-1 for 200 days, while Chlorella sp. was acclimated in medium containing 2 mg Cu L-1 for 100 days. Changes in algal growth rates and copper and zinc tolerance were monitored using standard growth inhibition toxicity tests in minimal medium over 72 h. Neither of the two acclimated N. closterium cultures had increased zinc or copper tolerance compared with that of the nonacclimated algae, nor were there any changes in control growth rates. Similarly, no changes in copper tolerance or control growth rates were observed for the acclimated Chlorella sp. culture. This was supported by measurements of intracellular and extracellular copper which confirmed that there were no differences in copper accumulation in either acclimated or nonacclimated algae. These results suggest that these algae grown in standard culture media are generally no more sensitive than algae grown in a metal-enriched medium. This supports the continued use of current laboratory bioassays with microalgae, as part of a suite of tests for assessing metal bioavailability, for use in ecological risk assessments and for providing data for the derivation of water quality guidelines. (c) 2007 Wiley Periodicals, Inc.

Kamiya E, Izumiyama S, Nishimura M, Mitchell JG, Kogure K (2007) Effects of fixation and storage on flow cytometric analysis of marine bacteria. Journal of Oceanography 63 :101-112

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Flow cytometry (FCM) is now becoming a routine tool for the enumeration and optical characterization of bacteria in marine environments. We investigated the effects of sample fixation and storage upon flow cytometric determination of marine bacteria. Fixed and unfixed seawater samples were analyzed by FCM immediately aboard ship and/or later in the laboratory, and the appearances of the fluorescence signals and bacterial counts of these samples were compared. Fixation and storage led to the formation of multiple peaks in fluorescence histograms ; this was also seen in 22 out of 36 samples frozen in liquid nitrogen. Fixation did not, but storage did induce a decrease of bacterial counts : a rapid decrease during the first 3 days followed by a slower decline. The decline of cell numbers in stored samples was expressed by a regression model. Our studies indicate that precaution is necessary when interpreting the data from fixed and/or stored marine bacterial samples analyzed by FCM. The possibility that the procedure of fixation and storage leads to the appearance of high DNA and low DNA bacterial groups should be considered.

Karshafian R, Bevan PD, Czarnota GJ, Burns PN, Samac S (2007) The dependence of sonoporation on cell cycle phase : Enhanced effect during G2 and S-phase. 2007 Ieee Ultrasonics Symposium Proceedings, Vols 1-6 :2005-2008

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Microbubble-assisted sonoporation, the ability to change local tissue permeability with ultrasound, which can promote the delivery of drugs and genes into cells, has been investigated both in vitro and in vivo. Under the same experimental and acoustic conditions, cells of the same histological type may be permeabilized, killed or left unaffected. We hypothesize that this variability depends partly on the cell-cycle phase. Here we investigate the effect of the cell-cycle on cell membrane permeability and viability in an in vitro cell suspension model using fluorescent markers and flow cytometry. Marine fibrosarcoma KHT-C cells in suspension were exposed to varying ultrasound conditions in the presence of Definity microbubbles. Cells were exposed to low (Pneg=125kPa) and high (Pneg=570kPa) acoustic pressures at 500kHz frequency, 16 cycles and 3kHz pulse repetition frequency in the presence of 3.5% volume concentration of microbubbles. Cell permeability and viability were measured for different phases of the cell-cycle (G1, S and G2) using flow cytometry. FITC-dextran was used to measure cell membrane permeability and Propidium Iodide was used to detect non-viable cells. Hoechst 33342 fluorescent marker was used to determine each cell’s cell-cycle phase. Data indicated that cells in different cell-cycle phases were permeabilized in different proportions. Cell permeability was 0.5%,25% and 45%, and cell viability was 78.8%,70.2% and 48.7% in untreated, 125 kPa-treated, and 570 kPa-treated samples, respectively. In the untreated sample, cell-cycle dependent viability after handling was 53.8% (G1), 42.6% (S) and 3.6% (G2) (normalized with respect to a total viability of 78.8%). In 125kPa-treated samples, cell viability was 70.6% (G1), 26.2% (S) and 1.7% (G2) (normalized to a total viability of 70.2%). Cell permeability was 20% (G1), 15% (S) and 2% (G2). The number of permeabilized to viable cells, was 0.23, 0.4 and 0.55 in G1, S and G2 phases, respectively (Pneg=125kPa). Cells in S and G2 were more susceptible to ultrasound compared to cells in G1 phase. Cells treated at 570kPa demonstrated a similar distribution in cell viability (70.8% (G1), 26.2% (S) and 2% (G2), normalized to total viability 48.7%), however, the distribution of cell permeability with respect to cell-cycle phase was different. In conclusion, cells in different cell-cycle phases demonstrated varied sensitivity to permeabilization when exposed to ultrasound and microbubbles. More cells were permeabilized and killed in S and G2 phases of their cell cycle. This may be related to differing cellular visco-elastic properties of the cell membrane that change during the cell cycle.

Katano T, Kaneda A, Kanzaki N, Obayashi Y, Morimoto A, Onitsuka G, Yasuda H, Mizutani S, Kon Y, Hata K, Takeoka H, Nakan S (2007) Distribution of prokaryotic picophytoplankton from Seto Inland Sea to the Kuroshio region, with special reference to ’Kyucho’ events. Aquatic Microbial Ecology 46 :191-201

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A ’Kyucho’ (an intrusion of warm surface water) occurs in the Bungo Channel, located in southwestern Japan. The abundances of Prochlorococcus and Synechococcus during a Kyucho, together with physical and chemical environmental factors, were investigated from the Kuroshio region to the Seto Inland Sea, via Bungo Channel, from 26 November to 5 December 2003. With the occurrence of the Kyucho, oceanic water intruded from the Kuroshio region into the middle of the Bungo Channel. The abundance of Prochlorococcus was the highest in the Kuroshio region and the southern part of the Bungo Channel (> 25 x 10(3) cells ml(-1)), low in the northern part of the Bungo Channel (< 1 x 10(3) cells ml(-1)), and below detection levels in the Seto Inland Sea. A relatively high abundance of Synechococcus cells (> 15 x 10(3) cells ml(-1)) was detected in the Kuroshio region and in the southern part of the Bungo Channel, but the abundance (< 6 x 10(3) cells ml(-1)) was low in other regions. In the Kuroshio region and the southern part of the Bungo Channel, high-phycourobilin (PUB)-type cells were dominant (> 90%) ; at this location, most of the available light in the deeper layer (> 25 m depth) was in the 450 to 500 nm range, corresponding to the peak absorbance of PUB. In contrast, the abundance of low-PUB-type cells accounted for > 75% of the total in the northern part of the Bungo Channel and in the Seto Inland Sea, where most of the available light in the deeper layer (> 10 m depth) was in the 480 to 560 nm range, including the peak absorbance of both PUB and phycoerythrobilin (PEB). These results indicate that Synechococcus cells of high-PUB type, which have a higher Ex 495:545 (> 1.5 ; ratio of orange fluorescence intensity excited at 495 nm to that at 545 nm), as well as Prochlorococcus cells were advected to the Bungo Channel by the Kyucho. The co-occurrence of the 2 pigment types of Synechococcus in coastal waters is highly affected by a physical process, such as the Kyucho.

Lambert C, Soudant P, Degremont L, Delaporte M, Moal J, Boudry P, Jean F, Huvet A, Samain JF (2007) Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites. Aquaculture 270 :276-288

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High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south : Baie des Veys (Normandy), Riviere d’Auray (Brittany) and Ronce (Marennes-oleron Basin, Poitou Charentes).
Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production.
Among the six families analyzed, three have shown high survival during summer (named as "resistant", mean mortality 5.2%) and three experienced high mortality during summer (named as "susceptible", 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic vibrio sp. S322 (50 bacteria/ hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date ; other relevant indicators would probably help explaining oyster survival during summer mortality events. (c) 2007 Elsevier B.V. All rights reserved.

Lambert C, Soudant P, Jegaden M, Delaporte M, Labreuche Y, Moal J, Samain JF (2007) In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes. Aquaculture 270 :413-421

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Bivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by Zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/ RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules ; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNl production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters. (c) 2007 Elsevier B.V. All rights reserved.

Levin M, Morsey B, De Guise S (2007) Modulation of the respiratory burst by organochlorine mixtures in marine mammals, humans, and mice. J Toxicol Environ Health A 70 :73-83


The effects of organochlorines (OC) on the immune systems of marine mammals and humans are poorly understood. One important innate immune function of peripheral blood neutrophils and monocytes is the respiratory burst, which generates reactive oxygen species (ROS) used to kill engulfed microorganisms. The present study characterized the immunomodulatory potential for mixtures of OCs, compared to that of individual OCs, on the respiratory burst in several marine mammals, humans, and B6C3F1 mice. The effects of three non-coplanar polychlorinated biphenyls (PCBs) (138, 153, 180), one coplanar PCB (169), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and all possible mixtures were tested upon in vitro exposure for 1 h, and their effects on the generation of a respiratory burst were measured by flow cytometry. The final concentration for each congener, alone or in a mixture, was 5 ppm for PCBs and 0.05 ppb for TCDD. Both significant enhancement and suppression of the respiratory burst occurred in all species tested, but the pattern was different between species and cell types (neutrophils vs. monocytes). Both coplanar and non-coplanar OCs were involved in the modulation of the respiratory burst. Regression analysis was not able to elucidate which OCs were involved in modulating the responses, highlighting the difficulty of developing models to predict the immunotoxic effects attributed to OC mixtures. The traditional mouse model and toxic equivalency (TEQ) approach both failed to consistently predict the toxicity of OCs in all species tested, questioning their applicability in the risk assessment process for all species. Elucidating the relative sensitivities to the immunomodulatory effects of OC mixtures between different species may have important implications for risk assessment as well as conservation and management strategies.

Levin M, Morsey B, De Guise S (2007) Non-coplanar PCBs induce calcium mobilization in bottlenose dolphin and beluga whale, but not in mouse leukocytes. J Toxicol Environ Health A 70 :1220-1231


Polychlorinated biphenyls (PCBs) have been demonstrated to modulate marine mammal immune functions ; however, the underlying mechanisms involved are poorly understood. Cytosolic calcium (Ca2+) is an important second messenger involved in numerous leukocyte functions. The direct effects of in vitro exposure to PCBs on Ca2+ mobilization were evaluated in leukocytes isolated from bottlenose dolphins, beluga whales, and B6C3F1 mice. Concentration- and time-response experiments with three non-coplanar PCBs (138, 153, 180), one coplanar PCB (169), and TCDD were tested. Exposure to the three non-coplanar PCBs significantly increased cytosolic Ca2+ in dolphin neutrophils, while PCB 180 significantly increased cytosolic Ca2+ in beluga neutrophils. Two non-coplanar PCBs (138 and 153) significantly increased Ca2+ in beluga monocytes, yet the response was delayed compared to that in neutrophils. Neither PCBs nor TCDD increased cytosolic Ca2+ in mouse neutrophils or monocytes. In experiments with Ca2+-free medium, only PCB 153 increased cytosolic Ca2+ in dolphin neutrophils, though the increase was less than that observed with Ca2+-supplemented medium, suggesting that extracellular Ca2+ was the predominant source for the rise in cytosolic Ca2+. Furthermore, in cells incubated with Ca2+-free medium, a significant increase in cytosolic Ca2+ was induced by thapsigargin following PCB exposure, indicating that intracellular Ca2+ was available, yet not mobilized by the PCBs, and further suggesting that PCBs mobilize extracellular Ca2+. These results demonstrate for the first time the direct effects of non-coplanar PCBs on Ca2+ mobilization in marine mammals, which may be involved in the modulation of phagocytosis previously observed in these species.

Levy JL, Stauber JL, Jolley DF (2007) Sensitivity of marine microalgae to copper : The effect of biotic factors on copper adsorption and toxicity. Science of the Total Environment 387 :141-154

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Microalgae are sensitive indicators of environmental change and, as the basis of most freshwater and marine ecosystems, are widely used in the assessment of risk and development of environmental regulations for metals. However, interspecies differences in sensitivity to metals are not well understood. The relationship between metal-algal cell binding and copper sensitivity of marine microalgae was investigated using a series of 72-h growth-rate inhibition bioassays and short-term (1-h) uptake studies. A range of marine algae from different taxonomic groups were screened to determine whether copper adsorption to the cell membrane was influenced by biotic factors, such as the ultrastructure of cell walls and cell size. Minutocellus polymorphus was the most sensitive species to copper and Dunaliella tertiolecta the least sensitive, with 72-h IC50 values (concentration to inhibit growth-rate by 50%) of 0.6 and 530 mu g Cu/L, respectively. Copper solution-cell partition coefficients at equilibrium (K-d) were calculated for six species of algae on a per cell and surface area basis. The largest and smallest cells had the lowest and highest Kd values, respectively (on a surface area basis), with a general (non-linear) trend of decreasing K-d with increasing cell surface area (p=0.026), however, no relationship was found between Kd and copper sensitivity, nor cell size and copper sensitivity. Interspecies differences in copper sensitivity were not related to cell size, cell wall type, taxonomic group or K-d values. The differences in sensitivity may be due to differences in uptake rates across the plasma membrane, in internal binding mechanisms and/or detoxification mechanisms between the different microalgal species. (C) 2007 Elsevier B.V. All rights reserved.

Li JL, Liu N, Chen XH, Sun M, Wang CB (2007) Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from Chlamys farreri in human HaCaT keratinocytes. Radiat Environ Biophys 46 :263-268


Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H(2)O(2)-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.

Liu YQ, Yao TD, Kang SC, Jiao NZ, Zeng YH, Huang SJ, Luo TW (2007) Microbial community structure in major habitats above 6000 m on Mount Everest. Chinese Science Bulletin 52 :2350-2357

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Bacterial abundance in surface snow between 6600 and 8000 m a.s.l. on the northern slope of Mt. Everest was investigated by flow cytometry. Bacterial diversity in serac ice at 6000 m a.s.l., glacier meltwater at 6350 m, and surface snow at 6600 m a.s.l. was examined by constructing a 16S rRNA gene clone library. Bacterial abundance in snow was higher than that in the Antarctic but similar to other mountain regions in the world. Bacterial abundance in surface snow increased with altitude but showed no correlation with chemical parameters. Bacteria in the cryosphere on Mt. Everest were closely related to those isolated from soil, aquatic environments, plants, animals, humans and,other frozen environments. Bacterial community structures in major habitats above 6000 m were variable. The Cytophaga-Flavobacterium-Bacteroides (CFB) group absolutely dominated in glacial meltwater, while beta-Proteobacteria and the CFB group dominated in serac ice, and beta-Proteobacteria and Actinobacteria dominated in surface snow. The remarkable differences among the habitats were most likely due to the bacterial post-deposition changes during acclimation processes.

Lohr J, Munn CB, Wilson WH (2007) Characterization of a latent virus-like infection of symbiotic zooxanthellae. Appl Environ Microbiol 73 :2976-2981


A latent virus-like agent, which we designated zooxanthella filamentous virus 1 (ZFV1), was isolated from Symbiodinium sp. strain CCMP 2465 and characterized. Transmission electron microscopy and analytical flow cytometry revealed the presence of a new group of distinctive filamentous virus-like particles after exposure of the zooxanthellae to UV light. Examination of thin sections of the zooxanthellae revealed the formation and proliferation of filamentous virus-like particles in the UV-induced cells. Assessment of Symbiodinium sp. cultures was used here as a model to show the effects of UV irradiance and induction of potential latent viruses. The unique host-virus system described here provides insight into the role of latent infections in zooxanthellae through environmentally regulated viral induction mechanisms.

Losekann T, Knittel K, Nadalig T, Fuchs B, Niemann H, Boetius A, Amann R (2007) Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby Mud Volcano, Barents Sea. Appl Environ Microbiol 73 :3348-3362


Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56%+/-8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94%+/-2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.

Lovoll M, Fischer U, Mathisen GS, Bogwald J, Ototake M, Dalmo RA (2007) The C3 subtypes are differentially regulated after immunostimulation in rainbow trout, but head kidney macrophages do not contribute to C3 transcription. Vet Immunol Immunopathol 117 :284-295


The complement system plays key roles in innate and adaptive immunity through mediating phagocytosis, chemotaxis and cell lysis. Complement component C3 is a central component in the complement cascade and belongs to the acute-phase proteins whose synthesis increase immediately upon inflammatory stimuli. The liver is the main producer of C3 and it is a well-known fact that the mammalian monocyte-macrophage lineage is a major contributor to extrahepatic C3. Immunomodulators, such as LPS and beta-glucan, can stimulate complement, lysozyme, natural killer cells and antibody responses in fish, thus enhancing the resistance to bacterial pathogens and parasitic infections. The aim of this study was to assess the effects of LPS and beta-glucan on the expression of interleukins (IL-1beta1, IL-1beta2 and IL-6) and the modulated expression of C3 subtypes (C3-1, C3-3 and C3-4) in the rainbow trout (Oncorhynchus mykiss) using real-time RT-PCR. From in vitro studies, we demonstrated that head kidney macrophages from rainbow trout and Atlantic salmon showed no basal transcription of C3. After immunostimulation, the cells responded by increased levels of ILs, but transcription of C3 was not induced. In contrast to the in vitro findings, the rainbow trout complement C3 subtypes were differentially regulated 48 h after in vivo stimulation with LPS and beta-glucan. These results support the previous findings of absence of C3 in macrophages of the spotted wolffish (Anarhichas minor) and is the first functional study showing differential regulation of the C3 subtypes in any vertebrate.

Martinez JM, Schroeder DC, Larsen A, Bratbak G, Wilson WH (2007) Molecular dynamics of Emiliania huxleyi and cooccurring viruses during two separate mesocosm studies. Appl Environ Microbiol 73 :554-562


In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi. Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi-dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.

Michelou VK, Cottrell MT, Kirchman DL (2007) Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic ocean. Appl Environ Microbiol 73 :5539-5546


We examined the contribution of photoheterotrophic microbes—those capable of light-mediated assimilation of organic compounds—to bacterial production and amino acid assimilation along a transect from Florida to Iceland from 28 May to 9 July 2005. Bacterial production (leucine incorporation at a 20 nM final concentration) was on average 30% higher in light than in dark-incubated samples, but the effect varied greatly (3% to 60%). To further characterize this light effect, we examined the abundance of potential photoheterotrophs and measured their contribution to bacterial production and amino acid assimilation (0.5 nM addition) using flow cytometry. Prochlorococcus and Synechococcus were abundant in surface waters where light-dependent leucine incorporation was observed, whereas aerobic anoxygenic phototrophic bacteria were abundant but did not correlate with the light effect. The per-cell assimilation rates of Prochlorococcus and Synechococcus were comparable to or higher than those of other prokaryotes, especially in the light. Picoeukaryotes also took up leucine (20 nM) and other amino acids (0.5 nM), but rates normalized to biovolume were much lower than those of prokaryotes. Prochlorococcus was responsible for 80% of light-stimulated bacterial production and amino acid assimilation in surface waters south of the Azores, while Synechococcus accounted for on average 12% of total assimilation. However, nearly 40% of the light-stimulated leucine assimilation was not accounted for by these groups, suggesting that assimilation by other microbes is also affected by light. Our results clarify the contribution of cyanobacteria to photoheterotrophy and highlight the potential role of other photoheterotrophs in biomass production and dissolved-organic-matter assimilation.

Moore LR, Coe A, Zinser ER, Saito MA, Sullivan MB, Lindell D, Frois-Moniz K, Waterbury J, Chisholm SW (2007) Culturing the marine cyanobacterium Prochlorococcus. Limnology and Oceanography-Methods 5 :353-362

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Prochlorococcus is the numerically dominant primary producer in open ocean ecosystems. Analysis of Prochlorococcus genome sequences from cultured isolates and ocean samples has broadened interest in studying this tiny cell, and efforts are underway to develop it into a model system for studying marine microbial ecology. A critical component of these efforts has been the development of systematic culturing methods that will facilitate the distribution of Prochlorococcus to diverse labs that may be interested in studying it. This paper provides detailed methods for maintaining cultures of Prochlorococcus, including a comparison of growth rates of cells on two artificial seawater media and on a standard medium that uses a natural seawater base. Procedures for agar plating, cryopreservation, obtaining new isolates, and issues associated with culture volume and carbon limitation also are described.

Moran XAG, Bode A, Suarez LA, Nogueira E (2007) Assessing the relevance of nucleic acid content as an indicator of marine bacterial activity. Aquatic Microbial Ecology 46 :141-152

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Current flow cytometry techniques allow the rapid estimation of the abundance of 2 distinct groups of heterotrophic bacteria, characterized by their relative nucleic acid content. High nucleic acid (HNA) bacteria are, at least in coastal environments, usually regarded as more active than the low nucleic acid (LNA) group. We tested the effects of substrate supply and bacterial cell size on the relationship between bacterial activity and the abundance of HNA bacteria by simultaneous measurements of LNA and HNA cell distributions, chlorophyll a and 3 H-leucine uptake rates in temperate shelf waters of the northern Iberian Peninsula. We considered 3 zones based on hydrological properties. Significant correlations were found between bacterial activity (range 0.1 to 80 pmol Leu l(-1) h(-1)) and both total and relative (range 28 to 84%) HNA cell abundance for pooled data, but the ready use of HNA bacterial abundance as a proxy for activity in natural systems was questioned by the low percentage of variance explained (16%). However, a detailed regional study of bottom-up effects revealed that the strength of this relationship increased significantly when bacteria were apparently controlled by phytoplankton substrate supply. Moreover, the relationship between mean biomass (overall range 12.4 to 21.2 fg C cell(-1)) and abundance-activity correlation coefficients in the 3 zones (r = 0.94, p = 0.005, n = 6) suggests that only at large cell sizes can we expect bacterial activity and production to be reasonably predicted by the abundance of HNA cells.

Nakayama A, Kurokawa Y, Harino H, Kawahara E, Miyadai T, Seikai T, Kawai S (2007) Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus). Aquatic Toxicology 83 :126-133

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Effects of tributyltin (TBT) which has been used for antifouling paint of ship’s hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out.
The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 mu g/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0 +/- 6.8 and 52.5 +/- 6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 mu g/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT.
To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated. (c) 2007 Elsevier B.V. All rights reserved.

Nancharaiah YV, Rajadurai M, Venugopalan VP (2007) Single cell level microalgal ecotoxicity assessment by confocal microscopy and digital image analysis. Environmental Science & Technology 41 :2617-2621

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In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dose-dependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDA technique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multi-parameter bioassay using confocal microscopy.

Not F, Valentin K, Romari K, Lovejoy C, Massana R, Tobe K, Vaulot D, Medlin LK (2007) Picobiliphytes : a marine picoplanktonic algal group with unknown affinities to other eukaryotes. Science 315 :253-255


Environmental sequencing has revealed unimagined diversity among eukaryotic picoplankton. A distinct picoplanktonic algal group, initially detected from 18S ribosomal DNA (rDNA) sequences, was hybridized with rRNA-targeted probes, detected by tyramide signal amplification-fluorescent in situ hybridization, and showed an organelle-like body with orange fluorescence indicative of phycobilins. Using this fluorescence signal, cells were sorted by flow cytometry and probed. Hybridized cells contained a 4’,6’-diamidino-2-phenylindole-stained organelle resembling a plastid with a nucleomorph. This suggests that they may be secondary endosymbiotic algae. Pending the isolation of living cells and their formal description, these algae have been termed picobiliphytes.

Orellana MV, Petersen TW, Diercks AH, Donohoe S, Verdugo P, van den Engh G (2007) Marine microgels : Optical and proteornic fingerprints. Marine Chemistry 105 :229-239

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Dissolved organic matter (DOM) is a major carbon reservoir for the global carbon cycle, and its molecules play a key role in the biogeochemistry of the ocean. Colloidal DOM macromolecules assemble to form polymer hydrogels known as marine microgels. Marine microgels represent one of the most dynamic pools of organic carbon in the ocean. However, their optical characteristics and their contribution to ocean optical properties are largely unknown. In this work, we explore the optical and proteomic properties of spontaneously assembled DOM polymer microgels. Microgels from cultures and from Puget Sound seawater were sorted and counted using a dual-laser (365 nm/365 nm) high-speed cell sorter. This sorter has been adapted to interface with a scanning monochromator to measure the fluorescence emission spectrum of the microgels over the range from 300 to 850 rim. Surprisingly, the microgels show a broad fluorescence emission from 420 to 520 nm when excited with UV light. The microgels were classified according to their blue autofluorescence, and by three criteria that are used to define microgels : 1) staining with chlortetracycline 2) the ability to undergo phase transitions at low pH, and 3) dispersion following calcium chelation by EDTA. (c) 2007 Elsevier B.V All rights reserved.

Pan LA, Zhang J, Zhang LH (2007) Picophytoplankton, nanophytoplankton, heterotrohpic bacteria and viruses in the Changjiang Estuary and adjacent coastal waters. Journal of Plankton Research 29 :187-197

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Flow cytometry (FCM) was used to examine the abundances and distributions of different picophytoplankton groups (i.e. Synechococcus, Prochlorococcus and picoeukaryotes), nanophytoplankton, heterotrophic bacteria and viruses were examined in the Changjiang Estuary, China and adjacent coastal waters during autumn 2004. Water temperature and light availability were found to be critical factors for picophytoplankton growth. Positive correlations were found between picophytoplankton, heterotrophic bacteria and viruses, and a seaward-increasing trend in the V-I (the group yielding high green fluorescence according to FCM) population within viruses was detected. The importance of nanophytoplankton is progressively usurped by picophytoplankton with increasing distance offshore. Picoeukaryotes are the most successful group among picophytoplankton in near-shore eutrophic waters, whereas Prochlorococcus surpasses other groups within the pico- and nanophytoplankton community in offshore oligotrophic regions of the East China Sea Shelf.

Parada V, Sintes E, van Aken HM, Weinbauer MG, Herndl GJ (2007) Viral abundance, decay, and diversity in the meso- and bathypelagic waters of the North Atlantic. Applied and Environmental Microbiology 73 :4429-4438

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To elucidate the potential importance of deep-water viruses in controlling the meso- and bathypelagic picoplankton community, the abundance, decay rate, and diversity of the virioplankton community were determined in the meso- and bathypelagic water masses of the eastern part of the subtropical North Atlantic. Viral abundance averaged 1.4 x 10(6) ml(-1) at around 100 m of depth and decreased only by a factor of 2 at 3,000 to 4,000 m of depth. In contrast, picoplankton abundance decreased by I order of magnitude to the Lower Deep Water (LDW ; 3,500- to 5,000-m depth). The virus- to-picoplankton ratio increased from 9 at about 100 m of depth to 110 in the LDW. Mean viral decay rates were 3.5 x 10(-3) h(-1) between 900 m and 2,750 m and 1.1 x 10(-3) h(-1) at 4,000 m of depth, corresponding to viral turnover times of 11 and 39 days, respectively. Pulsed-field gel electrophoresis fingerprints obtained from the viral community between 2,400 in and 4,000 in of depth revealed a maximum of only four bands from 4,000 in of depth. Based on the high viral abundance and the low picoplankton production determined via leucine incorporation, we conclude that the viral production calculated from the viral decay is insufficient to maintain the high viral abundance in the deep North Atlantic. Rather, we propose that substantial allochthonous viral input or lysogenic or pseudolysogenic production is required to maintain the high viral abundance detected in the meso- and bathypelagic North Atlantic. Consequently, deep-water prokaryotes are apparently far less controlled in their abundance and taxon richness by lytic prokaryotic phages than the high viral abundance and the virus-to-picoplankton ratio would suggest.

Park MO, Ikenaga H, Watanabe K (2007) Phage diversity in a methanogenic digester. Microbial Ecology 53 :98-103

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It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day(-1) L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

Patel A, Noble RT, Steele JA, Schwalbach MS, Hewson I, Fuhrman JA (2007) Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nature Protocols 2 :269-276

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Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20 - 200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes - Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-mu m aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.

Paul J, Scholin C, Van Den Engh G, Perry MJ (2007) In Situ Instrumentation. Oceanography 20 :70-78

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Ribalet F, Berges JA, Ianora A, Casotti R (2007) Growth inhibition of cultured marine. phytoplankton by toxic algal-derived polyunsaturated aldehydes. Aquatic Toxicology 85 :219-227

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Several marine diatoms produce polyunsaturated aldehydes (PUAs) that have been shown to be toxic to a wide variety of model organisms, from bacteria to invertebrates. However, very little information is available on their effect on phytoplankton. Here, we expand previous studies to six species of marine phytoplankton, belonging to different taxonomic groups that are well represented in marine plankton. The effect of three PUAs, 2E,4E-decadienal, 2EAE-octadienal and 2E,4E-heptadienal, was assessed on growth, cell membrane permeability, flow cytometric properties and morphology. A concentration-dependent reduction in the growth rate was observed for all cultures exposed to PUAs with longer-chained aldehydes having stronger effects on growth than shorter-chained aldehydes. Clear differences were observed among the different species. The prymnesiophyte Isochrysis galbana was the most sensitive species to PUA exposure with a lower threshold for an observed effect triggered by mean concentrations of 0.10 mu mol L-1 for 2E,4E-decadienal, 1.86 mu mol L-1 for 2E,4E-octadienal and 3.06 mu mol L-1 for 2E,4E-heptadienal, and a 50% growth inhibition (EC50) with respect to the control at 0.99, 2.25 and 5.90 mu mol L-1 for the three PUAs, respectively. Alternatively, the chlorophyte Tetraselmis suecica and the diatom Skeletonema marinoi (formerly S. costatum) were the most resistant species with 50% growth inhibition occurring at concentrations at least two to three times higher than L galbana. In all species, the three PUAs caused changes in flow cytometric measures of cell size and cell granulosity and increased membrane permeability, assessed using the viability stain SYTOX Green. For example, after 48 h 51.6 +/- .6% of I. galbana cells and 15.0 +/- 1.8% of S. marinoi cells were not viable. Chromatin fragmentation was observed in the dinoflagellate Amphidinium carterae while clear DNA degradation was observed in the chlorophyte Dunaliella tertiolecta. Concentrations used are in a significant range for affecting growth and performance of phytoplankton living in close vicinity of PUA-producing algae. Thus, PUAs may act as allelochemicals by mediating interactions among planktonic organisms. (c) 2007 Elsevier B.V. All rights reserved.

Ribalet F, Wichard T, Pohnert G, Ianora A, Miralto A, Casotti R (2007) Age and nutrient limitation enhance polyunsaturated aldehyde production in marine diatoms. Phytochemistry 68 :2059-2067

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Skeletonema marinoi produces 2,4-heptadienal, 2,4-octadienal, and 2,4,7-octatrienal, the latter only in traces. In nutrient-replete cultures, the production of potentially defensive polyunsaturated aldehydes (PUA) increases from the exponential to the stationary phase of growth from 1.2 fmol cell(-1) (+/- 0.4 fmol cell(-1) SD) to 4.2 fmol cell(-1) (+/- 1.0 fmol cell(-1) SD), with 2,4-heptadienal as the dominant aldehyde. The plasticity of PUA production with age of the culture supports the hypothesis of a direct link between toxin production and cell physiological state. N- and P-limited cells in stationary phase produced 1.4 and 1.8 fold higher amounts of PUA than control cultures and 10.7 and 4.6 times higher PUAs when compared to their own exponential growth phase, respectively. The increase in PUA production in the nutrient-limited cultures was not paralleled by an increase in the total amount of precursor fatty acids indicating that physiological stress might trigger an enhanced expression or activity of the enzymes responsible for PUA production, i.e. chemical defense increase in aged and nutrient-stressed diatoms. If this holds true during blooms, grazers feeding at the end of a bloom would be more affected than early-bloom grazers. (c) 2007 Elsevier Ltd. All rights reserved.

Russo J, Lefeuvre-Orfila L, Lagadic L (2007) Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environmental Pollution 146 :420-427

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Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 mu g/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed. (c) 2006 Elsevier Ltd. All rights reserved.

Salcher MM, Hofer J, Hornak K, Jezbera J, Sonntag B, Vrba J, Simek K, Posch T (2007) Modulation of microbial predator-prey dynamics by phosphorus availability : Growth patterns and survival strategies of bacterial phylogenetic clades. Fems Microbiology Ecology 60 :40-50

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We simultaneously studied the impact of top-down (protistan grazing) and bottom-up (phosphorus availability) factors on the numbers and biomasses of bacteria from various phylogenetic lineages, and on their growth and activity parameters in the oligo-mesotrophic Piburger See, Austria. Enhanced grazing resulted in decreased proportions of bacteria with high nucleic acid content (high-NA bacteria) and lower detection rates by FISH. There was a change in the composition of the bacterial assemblage, whereby Betaproteobacteria were heavily grazed while Alphaproteobacteria and Cytophaga-Flavobacterium-Bacteroides were less affected by predators. Changes in bacterial assemblage composition were also apparent in the treatments enriched with phosphorus, and even more pronounced in the incubations in dialysis tubes (allowing relatively free nutrient exchange). Here, Betaproteobacteria became dominant and appeared to act as successful opportunistic competitors for nutrients. In contrast, Actinobacteria did not respond to surplus phosphorus by population growth, and, moreover, maintained their small size, which resulted in a very low biomass contribution. In addition, significant relationships between high-NA bacteria and several bacterial phylogenetic clades were found, indicating an enhanced activity status. By combining several single-cell methods, new insight is gained into the competitive abilities of freshwater bacteria from a variety of phylogenetic lineages under contrasting sets of bottom-up and top-down constraints.

Santic D, Krstulovic N, Solic M (2007) Comparison of flow cytometric and epifluorescent counting methods for marine heterotrophic bacteria. Acta Adriatica 48 :107-114

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Two direct heterotrophic bacterioplankton counting methods, epifluorescence microscopy (EM) and flow cytometry (FCM) were compared using samples collected in two geographically different oceanic regions, the Adriatic Sea and the English Channel. A statistically significant correlation was found between results obtained by these two methods for samples collected in the Adriatic Sea (r =0. 61, n =919, P <0.001) and in the English Channel (r =0. 64, n =33, P <0. 001). Samples from the Adriatic Sea showed on average 1.16 times higher values obtained by flow cytometry than values estimated by epifluorescence microscopy, while samples from the English Channel showed on average a 0.74 ratio between flow cytometry and epifluorescence microscopy counts. The overall coefficient of variation for epifluorescence microscopy data for samples from the Adriatic Sea and the English Channel was 15.91% and 12.89%, respectively. The flow cytometry method had lower overall coefficient of variation value ; for samples collected in the Adriatic Sea it was 3.64%, while for samples collected in the English Channel it was 1.88%.

Scharek R, Latasa M (2007) Growth, grazing and carbon flux of high and low nucleic acid bacteria differ in surface and deep chlorophyll maximum layers in the NW Mediterranean Sea. Aquatic Microbial Ecology 46 :153-161

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Growth and grazing mortality of marine heterotrophic bacteria were measured in the summer of 2000 in coastal waters of the NW Mediterranean Sea. Serial-dilution experiments were performed with water from surface and deep chlorophyll maximum (DCM) layers. Bacterial abundances (mean +/- SD) were very similar at the surface (7.2 +/- 2.9 x 10(5) cells ml(-1)) and DCM (7.4 +/- 1.1 x 105 cells ml-1). Intrinsic bacterial growth rates (mean +/- SD) were 0.88 +/- 0.43 d(-1) in the surface layer and 0.71 +/- 0.23 d(-1) at the DCM. Grazing rates on bacteria (mean +/- SD) were 0.75 +/- 0.23 and 0.58 +/- 0.29 d(-1), in the surface and DCM layers, respectively. Nucleic acid content analysis by flow cytometry revealed different intrinsic growth rates and grazing pressure on bacteria of high (HNA) and low (LNA) content depending on their location in the water column. Generally, growth and grazing rates were balanced in both groups in both layers. At the surface, HNA bacteria revealed significantly higher intrinsic growth rates than LNA bacteria (1.18 +/- 0.60 and 0.47 +/- 0.28 d(-1), respectively). Average growth rates at the DCM were higher for LNA (0.90 +/- 0.46 d(-1)) than for HNA (0.36 +/- 0.23 d(-1)), but not significantly. At the surface, grazing rates on HNA bacteria were also significantly higher than on LNA bacteria (1.02 +/- 0.31 and 0.37 +/- 0.19 d(-1), respectively). At the DCM, the opposing tendency, though not statistically significant, was observed (0.26 +/- 0.17 and 0.77 +/- 0.50 d(-1)). Bacteria were responsible for a large portion of the C flux through the system. Bacterial C flux was funneled mostly by HNA bacteria at the surface (70%) and by LNA at the DCM (80%). HNA bacteria were the most active component of the bacterial community in the surface layer. However, we found that LNA bacteria were also active, particularly at the DCM, suggesting differences in structure and functioning of the corresponding microbial networks. The most significant result was the clear relation between depth and activity of each bacterial fraction.

Seymour JR, Seuront L, Mitchell JG (2007) Microscale gradients of planktonic microbial communities above the sediment surface in a mangrove estuary. Estuarine Coastal and Shelf Science 73 :651-666

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The microscale (1 and 4 cm sampling resolution) distributions of chemical (O-2, NH3, NO3, NO2-, PO43-) and biological (Ch1 a, phytoplankton, bacterioplankton, viruses) parameters were measured in the 16 cm of water immediately overlaying the sediment-water interface (SWI) within a temperate mangrove estuary in South Australia during December 2003 and March 2004. Shear velocities (u(*)) during the time of sampling were very low (< 0.1 cm s(-1)), and we consequently predict that resuspension of organisms and materials was negligible. In December 2003, profiles were often characterised by strong gradients in nutrients and organisms, with the highest concentrations often observed within 0.5 cm of the SWI. Microscale patterns in O-2, NH3, NO3- and NO2- indicated that a variety of anaerobic and aerobic transformation processes probably occurred at the SWI and within profiles. Strong gradients in PO43- were indicative of nutrient flux across the SWI as a consequence of degradation processes in the sediments. Pico- and nanophytoplankton concentrations were strongly correlated (p < 0.01) to PO43-, and exhibited 12- and 68-fold changes in abundance, respectively, with highest concentrations observed nearest to the SWI. Several bacterial subpopulations were discriminated using flow cytometry and significant shifts in the ’cytometric structure’ of the bacterial community were observed within microscale profiles. Two populations of viruses were correlated to the phytoplankton and low DNA (LDNA) bacteria, and each exhibited elevated concentrations within 0.5 cm of the SWI. In March 2004, microscale distributions of O-2 and nutrients were more homogenous than in December 2003, and dissimilar microbial community structure and patterns were observed above the SWI. The patterns observed here support the prediction that benthic processes can strongly influence the ecology of planktonic communities in the overlaying water, and provide further evidence for the existence of microscale variability amongst communities of aquatic microorganisms. (c) 2007 Elsevier Ltd. All rights reserved.

Shang X, Zhang LH, Zhang J (2007) Prochlorococcus-like populations detected by flow cytometry in the fresh and brackish waters of the Changjiang Estuary. Journal of the Marine Biological Association of the United Kingdom 87 :643-648

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Pico-size cells with a red fluorescence signature similar to marine free-living prochlorophytes have been detected by flow cytometry throughout the fresh and brackish waters of the Changjiang Estuary in August 2005. These cells could be discriminated from Synechococcus by their lack of orange (phycoerythrin) fluorescence, from Prochlorococcus by their larger side light scatter, and from picoeukaryotes by their smaller side light scatter and much lower red (chlorophyll) fluorescence. The maximum abundances of these Prochlorococcus-like particles reached 3 x 10(4) cells ml(-1) in the low salinity zone upriver and declined rapidly seaward. While Synechococcus was scarce and Prochlorococcus vanished in the brackish regions, the Prochlo rococcus- like cells overnumbered the picoeukaryotes and became the predominant pico-size autofluorescing particles there, implying they might play an important role in this estuarine ecosystem and worthy of further investigation.

Shi XL, Kong FX, Yu Y, Yang Z (2007) Survival of Microcystis aeruginosa and Scenedesmus obliquus under dark anaerobic conditions. Marine and Freshwater Research 58 :634-639

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The cyanobacterium Microcystis aeruginosa and the green alga Scenedesmus obliquus were incubated individually and together in the dark and under anaerobic conditions created by adding the reducing agent cysteine. Flow cytometry was used to monitor cell concentrations, fluorescence of chlorophyll-a (chl-a), and cell metabolic activity measured with an esterase-sensitive probe to detect fluorescein diacetate (FDA) hydrolysis of the two species. M. aeruginosa showed a slight increase in cell metabolic activity, no conspicuous death of cells, and absence of decay of chlorophyll-a fluorescence in individual and competition cases under dark anaerobic conditions. Cell metabolic activity and fluorescence of S. obliquus, on the contrary, decreased sharply, and cell concentrations fluctuated markedly with time in the unialgal cultures, but showed only a slight decline in the mixed cultures. M. aeruginosa appeared to be more tolerant to dark anaerobic conditions than S. obliquus, which may arise in eutrophic lakes beneath thick surface scums in the water column, or in the bottom sediments. Tolerance of these conditions may be important to the dominance of M. aeruginosa in eutrophic lakes.

Siemieniuch M, Dubiel A (2007) Preservation of tomcat (Felis catus) semen in variable temperatures. Animal Reproduction Science 99 :135-144

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The aim of our study was to estimate the viability of cat epididymal sperm in short time storage at +4 degrees C and in long term storage at -196 degrees C and to assess the percentage of live sperm in fresh semen using eosin/nigrosin staining compared to the flow cytometry method.
The testes with epididymides were obtained after routine castration procedure. The sperm for further research were collected after flushing the epididymides using extender consist of : Tris 2.4 g, citric acid 1.4 g, glucose 0.8 g, 0.06% (w/v) Na-benzylpenicillin, 0.1% (w/v) streptomycin sulphate and distilled water. Half of each sample was equilibrated with the dilution and loaded in 0.25 ml plastic straws. The straws were placed on a rack in liquid nitrogen vapour at -120 degrees C for 10 min, plunged in liquid nitrogen for 10 min, replaced to marked goblets and loaded into canes for long term storage in liquid nitrogen at -196 degrees C. Sixty percent of motile spermatozoa was accomplished after thawing. However, the percentage of the sperm with intact acrosomes was decreased and the share of cells with midpiece and tail defects was increased. The storage of sperm flushed from epididymides at WC for a short time and the usage of sperm during 2-3 days after collection seems to be better than cryopreservation.
In our study, normospermia was present in 72.7 +/- 8.8% of fresh semen. The most common defect was the presence of distal droplets, imperfect heads or abnormal acrosomal outline. The motility of fresh sperm flushed from epididymides achieved 77.9 +/- 6.8%. The viability of sperm amounting to 52.5 +/- 13.8% was achieved on third day of conservation in the liquid extender.
The percentage of viable sperm in fresh epididymal spermatozoa was 84.9 +/- 7.8%. Compared to these results. the percentage of live cells using SYBR-14/propidium iodide staining was insignificantly lower (82.2 +/- 8%). The live, non-apoptotic cells were 79.0 +/- 7.8%. The share of live, early-apoptotic spermatozoa and late-apoptotic spermatozoa was, respectively, 2 +/- 1.4% and 1.5 +/- 0.9%. The viability of sperm estimated by eosin/nigrosin staining was confirmed by the flow cytometry method. There was no statistical differences between the staining, The usage of apoptosis detection kit revealed, that the percentage of early-apoptotic and late-apoptotic cells was insignificant. (c) 2006 Published by Elsevier B.V.

Simpson SL, Micevska T, Adam MS, Stone A, Maher WA (2007) Establishing cause-effect relationships in hydrocarbon-contaminated sediments using a sublethal response of the benthic marine alga, Entomoneis cf punctulata. Environ Toxicol Chem 26 :163-170


A sublethal whole-sediment toxicity test that uses flow cytometry to measure inhibition of esterase activity in the marine microalga Entomoneis cf punctulata was applied to the assessment of hydrocarbon-contaminated sediments and toxicity identification and evaluation (TIE). Concentration-response relationships were developed, and a 20% effect concentration for total polycyclic aromatic hydrocarbons (PAHs) of 60 mg/kg normalized to 1% total organic carbon was calculated. Relationships between toxic effects and sediment organic carbon concentrations, organic carbon forms (e.g., black carbon), and sediment particle size indicated that further normalization of hydrocarbon concentrations to sediment particle size may improve concentration-response relationships. The algal toxicity test was applied as a rapid whole-sediment TIE procedure that involved the addition to sediment of powdered coconut charcoal (PCC), a hydrophobic, carbon-based material that strongly adsorbs PAHs and decreases the pore-water exposure pathway. Sediments with PCC concentrations of up to 15% (w/w) provided acceptable responses in control sediments. For six sediments with total PAH concentrations of 1,060, 4,060, 5,120, 9,150, 9,900, and 15,900 mg/kg, inhibition of E. cf punctulata esterase activity (% of control) was 75, 97, 94, 93, 100, and 97%, respectively. Following a 15% PCC amendment to these sediments, inhibition of esterase activity was 0, 1, 11, 69, 32, and 68%, respectively, indicating a decrease in toxicity in all sediments. Because the alga E. cf punctulata is exposed to toxicants via both pore water and overlying water, the reduction in toxicity achieved by 15% PCC additions can be related to the efficient removal of dissolved hydrocarbons released from sediment particles. The sediment-PCC manipulations coupled with algal whole-sediment toxicity tests provided an effective and rapid TIE method to determine whether hydrocarbon contaminants are responsible for toxicity in sediments.

Stepanauskas R, Sieracki ME (2007) Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proceedings of the National Academy of Sciences of the United States of America 104 :9052-9057

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The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacleria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88-97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, Suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MIDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted <11% of the sampled bacterioplankton. Compared with metagenomics, the power of our approach lies in the ability to detect metabolic genes in uncultured microorganisms directly, even when the metabolic and phylogenetic markers are located far apart on the chromosome.

Stomp M, Huisman J, Voros L, Pick FR, Laamanen M, Haverkamp T, Stal LJ (2007) Colourful coexistence of red and green picocyanobacteria in lakes and seas. Ecology Letters 10 :290-298

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Hutchinson’s paradox of the plankton inspired many studies on the mechanisms of species coexistence. Recent laboratory experiments showed that partitioning of white light allows stable coexistence of red and green picocyanobacteria. Here, we investigate to what extent these laboratory findings can be extrapolated to natural waters. We predict from a parameterized competition model that the underwater light colour of lakes and seas provides ample opportunities for coexistence of red and green phytoplankton species. To test this prediction, we sampled picocyanobacteria of 70 aquatic ecosystems, ranging from clear blue oceans to turbid brown peat lakes. As predicted, red picocyanobacteria dominated in clear waters, whereas green picocyanobacteria dominated in turbid waters. We found widespread coexistence of red and green picocyanobacteria in waters of intermediate turbidity. These field data support the hypothesis that niche differentiation along the light spectrum promotes phytoplankton biodiversity, thus providing a colourful solution to the paradox of the plankton.

Sun P, Zhang X, Zang X, Zhou X, Chen Y, Arunakumara KK, Liang B (2007) Anti-hypercalcemic effect of orally administered recombinant Saccharomyces cerevisiae expressing salmon calcitonin on hypercalcemic rats. Biotechnol Lett 29 :1013-1018


Oral delivery of salmon calcitonin (sCT) to rats via a recombinant Saccharomyces cerevisiae was assessed. A synthetic sCT gene was cloned and expressed in S. cerevisiae yAGA2-sCT. Recombinant salmon calcitonin (rsCT) expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by 3 x 10(-6 )M rsCT. Oral administration of 5 g lyophilized yAGA2-sCT/kg to hypercalcemic rats decreased serum calcium from 2.8 +/- 0.02-2.7 +/- 0.02 mM.

Suttle CA (2007) Marine viruses - major players in the global ecosystem. Nature Reviews Microbiology 5 :801-812

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Viruses are by far the most abundant ` lifeforms’ in the oceans and are the reservoir of most of the genetic diversity in the sea. The estimated 1030 viruses in the ocean, if stretched end to end, would span farther than the nearest 60 galaxies. Every second, approximately 1023 viral infections occur in the ocean. These infections are a major source of mortality, and cause disease in a range of organisms, from shrimp to whales. As a result, viruses influence the composition of marine communities and are a major force behind biogeochemical cycles. Each infection has the potential to introduce new genetic information into an organism or progeny virus, thereby driving the evolution of both host and viral assemblages. Probing this vast reservoir of genetic and biological diversity continues to yield exciting discoveries.

Tang YZ, Dobbs FC (2007) Green autofluorescence in dinoflagellates, diatoms, and other microalgae and its implications for vital staining and morphological studies. Applied and Environmental Microbiology 73 :2306-2313

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Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships’ ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAIT in a broad phylogenetic range of microalgae. Our results demonstrate GAIT is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships’ ballast tanks.

Tavecchio M, Natoli C, Ubezio P, Erba E, D’Incalci M (2007) Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach. Cell Prolif 40 :885-904


OBJECTIVES : Trabectedin (ET-743, Yondelis) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)-rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug-induced cell cycle perturbations. MATERIALS AND METHODS : An isogenic NER-proficient cellular system (CHO-AA8) and a NER-deficient one (CHO-UV-96), lacking functional ERCC-1, were studied. Flow cytometric assays showed progressive accumulation of cells in G2 + M phase in NER-proficient but not in NER-deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. RESULTS : Cells exposed to trabectedin during G1 and G2 + M first experienced a G1 block, while those exposed in S phase were delayed in S and G2 + M phases but eventually divided. In the presence of functional NER, exit from the G1 block was faster ; then, cells progressed slowly through S phase and were subsequently blocked in G2 + M phase. This G2 + M processing of trabectedin-induced damage in NER-proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. CONCLUSIONS : This might be due either to important damage left unrepaired by previous G1 repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV-96 cells repair mechanisms other than NER are activated both in G1 and G2 + M phases.

Thomas P, Pang Y, Dong J, Groenen P, Kelder J, de Vlieg J, Zhu Y, Tubbs C (2007) Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins. Endocrinology 148 :705-718


A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR alpha subtypes from spotted seatrout (st-mPRalpha) and humans (hu-mPRalpha) encode progestin receptors that display many functional characteristics of G protein-coupled receptors. Flow cytometry and immunocytochemical staining of whole MDA-MB-231 cells stably transfected with the mPRalphas using antibodies directed against their N-terminal regions show the receptors are localized on the plasma membrane and suggest the N-terminal domain is extracellular. Both recombinant st-mPRalpha and hu-mPRalpha display high affinity (Kd 4.2-7.8 nm), limited capacity (Bmax 0.03-0.32 nm), and displaceable membrane binding specific for progestins. Progestins activate a pertussis toxin-sensitive inhibitory G protein (G(i)) to down-regulate membrane-bound adenylyl cyclase activity in both st-mPRalpha- and hu-mPRalpha-transfected cells. Coimmunoprecipitation experiments demonstrate the receptors are directly coupled to the G(i) protein. Similar to G protein-coupled receptors, dissociation of the receptor/G protein complex results in a decrease in ligand binding to the mPRalphas and mutation of the C-terminal, and third intracellular loop of st-mPRalpha causes loss of ligand-dependent G protein activation. Phylogenetic analysis indicates the mPRs are members of a progesterone and adipoQ receptor (PAQR) subfamily that is only present in chordates, whereas other PAQRs also occur in invertebrates and plants. Progesterone and adipoQ receptors are related to the hemolysin3 family and have origins in the Eubacteria. Thus, mPRs arose from Eubacteria independently from members of the GPCR superfamily, which arose from Archeabacteria, suggesting convergent evolution of seven-transmembrane hormone receptors coupled to G proteins.

Tiebre MS, Vanderhoeven S, Saad L, Mahy G (2007) Hybridization and sexual reproduction in the invasive alien Fallopia (Polygonaceae) complex in Belgium. Annals of Botany 99 :193-203

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Background and Aims The knotweed complex, Fallopia spp. (Polygonaceae), belongs to the most troublesome invasive species in Europe and North America. Vegetative regeneration is widely recognized as the main mode of reproduction in the adventive regions. However, the contribution of sexual reproduction to the success of these invasive species has only been detailed for the British Isles. An examination was made as to how hybridization may influence the sexual reproduction of the complex in Belgium and to determine how it may contribute to the dispersal of the species.
Methods Studies were made of floral biology, reproductive success, seed rain, seed bank, germination capacity, seedling survival and dispersal capacity in order to characterize the reproductive biology of the species. Moreover, chromosome counts and flow cytometry were used to assess the hybrid status of seedlings produced by sexual reproduction.
Key Results In the area investigated, extensive sexual reproduction by hybridization within the complex, including one horticultural species, was demonstrated. A small percentage of seeds may be dispersed outside the maternal clone (> 16 m) allowing the formation of genetically differentiated individuals. Seed germination was possible even after a winter cold period.
Conclusions The extensive sexual reproduction by hybridization could further contribute to the dramatic invasive success of knotweeds in Belgium and should not be underestimated when considering control and management measures.

Tomaru Y, Nagasaki K (2007) Flow cytometric detection and enumeration of DNA and RNA viruses infecting marine eukaryotic microalgae. Journal of Oceanography 63 :215-221

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Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized : Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large DNA algal viruses HaV and HcV (similar to 0.2 mu m in diameter) were similar to numbers determined by epifluorescence microscopy (EFM). In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community may be overlooked by FCNI.

Touzet N, Raine R (2007) Discrimination of Alexandrium andersoni and A-minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridization. Phycologia 46 :168-177

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Toxic marine dinoflagellates from the genus Alexandrium have been responsible for paralytic shellfish poisoning (PSP) throughout the world. Their monitoring relies on spatial and temporal sampling strategies and requires the reliable identification and enumeration of vegetative stages in order to enable the development of early warning policies. The accurate discrimination between Alexandrium species is tabour intensive and requires taxonomic expertise as the genus contains morphologically similar toxic and non-toxic species. In Ireland, PSP outbreaks so far have been limited to Cork Harbour, a retentive inlet located on the south coast of the country, where the causative organism has been identified as A. minutum. Recently, the non-toxic and morphologically similar species A. andersoni has been detected on the south west coast of Ireland. In routine monitoring, Alexandrium spp. are identified on the basis of morphological features by conventional light microscopy, a method which does not allow their characterization at the species level. The development and application of large subunit (LSU) rRNA-targeted oligonucleotide probes for the detection and quantification of A. minutum (Global clade) and A. andersoni by whole-cell fluorescent in situ hybridization (FISH) is reported. The specificity and sensitivity of the two probe sets (MinA and And A’+C) were evaluated against Alexandrium species, including A. tamarense, A. tamutum and A. ostenfeldi, and a range of common dinoflagellates usually co-occurring with Alexandrium in Irish coastal waters. No cross-reactivity was observed with any of the strains tested or with phytoplankton species present in field samples rich in dinoflagellates. The format of the assay overcame possible matrix effects, such as probe adsorption, and allowed the reliable labelling of at least 1000 cells. Furthermore, the simultaneous use of calcofluor during the assays permitted the confirmation of the probe diagnostics by examining the general plate structure of the thecae of labelled cells.

Unrein F, Massana R, Alonso-Saez L, Gasol JM (2007) Significant year-round effect of small mixotrophic flagellates on bacterioplankton in an oligotrophic coastal system. Limnology and Oceanography 52 :456-469

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The seasonal variation in the grazing effect of mixotrophic flagellates on bacterioplankton was assessed during an annual cycle in an oligotrophic coastal station in the northwest Mediterranean Sea. Ingestion rates of fluorescently labeled bacteria were estimated for different size categories of phytoflagellates (PF) and heterotrophic flagellates (HF) in short-te
rm experiments and compared with long-term grazing estimates and published empirical models. The mixotrophic flagellates included haptophyte-like cells, cryptophytes, and dinoflagellates. The group-specific grazing rates (SGR) averaged 1.1 (3-5 mu m PF), 1.3 (5-20 mu m PF), 4.0 (< 5 mu m HF), and 15.4 bacteria individual(-1) h(-1) (5-20 mu m HF). Lower SGR but higher abundances of PF resulted in an average mixotroph contribution of 50% to the total flagellate grazing. Remarkably, the effect was relatively high all through the year (35-65%). Regardless of the presence of chloroplasts, flagellates < 5 mu m in size accounted, on average, for about 80% of total flagellate bacterivory and ingested a large percentage of their cell carbon per day from bacteria. Soluble reactive phosphorus concentration was negatively correlated with the ingestion rate of both groups of PF, suggesting that mixotrophic flagellates would be using their phagotrophic capability to obtain phosphorus when this nutrient is limiting. HF grazing activity showed a marked seasonality, with grazing being higher during the warmer seasons, and clearance rates were positively correlated with water temperature. Total bacterivory accounted for most of the bacterial production. Short-term and long-term bacterivory measurements were highly correlated, confirming that the smallest flagellates were the main causative agent of bacterial loss. The bacterivory values were also well correlated to a published empirical model that considers HF as the only bacterivorous. However, this model underestimated (up to 50%) total flagellate grazing during periods of high effect of mixotrophic flagellates.

Veldhuis MJW, Timmermans KR (2007) Phytoplankton dynamics during an in situ iron enrichment experiment (EisenEx) in the Southern Ocean : a comparative study of field and bottle incubation measurements. Aquatic Microbial Ecology 47 :191-208

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Composition, physiology and growth response of 5 size classes of phytoplankton, ranging from 0.7 to 20 mu m, was studied in an in situ iron enrichment experiment (EisenEx) and in bottle incubations in the Southern Ocean during austral spring 2000. In the field, iron enrichment resulted in only minor changes in numerical abundance, cell carbon content, photosynthetic efficiency (F-v:F-m) and the percentage of live cells of Synechococcus spp. and pico-eukaryotes (<2 mu m). In these 2 groups, only cellular chlorophyll a (chl a) content increased (by 20 and 100%, respectively). The physiological conditions of the 2 to 20 pm cells improved significantly, but a statistically significant (3-fold higher) biomass was observed only in the largest size fraction (8 to 20 mu m). Bottle experiment results were comparable with in situ results, except that the responses occurred earlier. In addition, the total increase in biomass was much larger than that in the field (100- and 25-fold increase for cell carbon and chl a content, respectively). In this respect the control and the iron-enriched bottles showed a comparable trend, suggesting that, next to iron, light was probably a factor of great importance. In the field, phytoplankton cells experience rapidly changing light conditions due to wind-induced turbulence that causes mixing of the water column. In addition to iron-limitation, this results in a generally poor physiological condition of algae in the field. This light-stressor mainly affected the smallest algal size classes (< 8 mu m). In larger cells, iron enrichment partly compensated for this negative light effect, resulting in a final dominance of larger phytoplankton.

Vital M, Fuchslin HP, Hammes F, Egli T (2007) Growth of Vibrio cholerae O1 Ogawa Eltor in freshwater. Microbiology 153 :1993-2001


Growth of Vibrio cholerae O1 Ogawa Eltor was studied with a growth assay in which autoclaved and filtered (0.22 microm) freshwater was inoculated at low cell density (5 x 10(3) cells ml(-1)) and proliferation was followed with flow cytometry. Against the common view, V. cholerae was able to grow extensively in different kinds of freshwater. The bacterium multiplied in river water, lake water and effluent of a wastewater treatment plant up to a cell density of 1.55 x 10(6) cells ml(-1). In these samples, apparent assimilable organic carbon (AOC(app)) concentrations ranged from 52 up to 800 microg l(-1) and the results demonstrate a positive trend between the AOC(app) concentration and final cell concentration, suggesting that AOC was a key parameter governing growth of V. cholerae. No growth was observed in waters (tap and bottled drinking water) containing less than approximately 60 microg AOC(app) l(-1). When pure cultures of V. cholerae were grown on identical lake water at different temperatures (20, 25 and 30 degrees C) the maximum specific growth rates (micromax) achieved were 0.22 h(-1), 0.32 h(-1) and 0.45 h(-1), respectively. In addition, growth was characterized in lake water samples amended with different concentrations of NaCl. The highest micromax of V. cholerae was recorded at moderate salinity levels (5 g NaCl l(-1), micromax=0.84 h(-1)), whereas at 30 g NaCl l(-1) (micromax=0.30 h(-1)) or 0 g NaCl l(-1) (micromax)=0.40 h(-1)) specific growth rates were significantly reduced. In the water tested here, micro(max) of V. cholerae was always around 50 % of that exhibited by a freshwater community of indigenous bacteria enriched from the water sampling site. Direct batch competition experiments between V. cholerae and the lake water bacterial community were performed at different temperatures in which V. cholerae was enumerated in the total community using fluorescent-surface antibodies. In all cases V. cholerae was able to grow and constituted around 10 % of the final total cell concentration of the community. No significant effect of temperature was observed on the outcome of the competition. Mathematical modelling of the competition at the different temperatures based on the calculated micromax values confirmed these experimental observations. The results demonstrate that V. cholerae is not only able to survive, but also able to grow in freshwater samples. In these experiments the bacterium was able to use a large fraction (12-62 %) of the AOC(app) available to the bacterial AOC-test community, indicating that V. cholerae has the ability to gain access to the substrates present in freshwater even in competition with an autochthonous bacterial lake water consortium.

Vital M, Fuchslin HP, Hammes F, Egli T (2007) Growth of Vibro cholerae O1 Ogawa Eltor in freshwater. Microbiology-Sgm 153 :1993-2001

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Growth of Vibrio cholerae O1 Ogawa Eltor was studied with a growth assay in which autoclaved and filtered (0.22 mu m) freshwater was inoculated at low cell density (5x10(3) cells ml(-1)) and proliferation was followed with flow cytometry. Against the common view, V. cholerae was able to grow extensively in different kinds of freshwater. The bacterium multiplied in river water, lake water and effluent of a wastewater treatment plant up to a cell density of 1.55x10(6) cells ml(-1). In these samples, apparent assimilable organic carbon (AOC(app)) concentrations ranged from 52 up to 800 mu g l(-1) and the results demonstrate a positive trend between the AOC(app) concentration and final cell concentration, suggesting that AOC was a key parameter governing growth of V cholerae. No growth was observed in waters (tap and bottled drinking water) containing less than approximately 60 pg AOC(app) l(-1). When pure cultures of V. cholerae were grown on identical lake water at different temperatures (20, 25 and 30 degrees C) the maximum specific growth rates (mu(max)) achieved were 0.22 h(-1), 0.32 h(-1) and 0.45 h(-1), respectively. In addition, growth was characterized in lake water samples amended with different concentrations of NaCl. The highest mu(max) of V. cholerae was recorded at moderate salinity levels (5 g NaCl l(-1), mu(max)=0.84 h(-1)), whereas at 30 g NaCl l(-1) (mu(max) =0.30 h(-1)) or 0 g NaCl l(-1) (mu(max)=0.40 h(-1)) specific growth rates were significantly reduced. In the water tested here, mu(max) of V. cholerae was always around 50 % of that exhibited by a freshwater community of indigenous bacteria enriched from the water sampling site. Direct batch competition experiments between V. cholerae and the lake water bacterial community were performed at different temperatures in which V. cholerae was enumerated in the total community using fluorescent-surface antibodies. In all cases V cholerae was able to grow and constituted around 10 % of the final total cell concentration of the community. No significant effect of temperature was observed on the outcome of the competition. Mathematical modelling of the competition at the different temperatures based on the calculated mu(max) values confirmed these experimental observations. The results demonstrate that V. cholerae is not only able to survive, but also able to grow in freshwater samples. In these experiments the bacterium was able to use a large fraction (112-62 %) of the AOCapp available to the bacterial AOC-test community, indicating that V. cholerae has the ability to gain access to the substrates present in freshwater even in competition with an autochthonous bacterial lake water consortium.

Wang Y, Hammes F, Boon N, Egli T (2007) Quantification of the filterability of freshwater bacteria through 0.45, 0.22, and 0.1 microm pore size filters and shape-dependent enrichment of filterable bacterial communities. Environ Sci Technol 41 :7080-7086


Micro-filtration is a standard process for sterilization in scientific research, medical, and industrial applications, and to remove particles in drinking water or wastewater treatment. It is generally assumed, and confirmed by quantifying filtration efficiency by plating, that filters with a 0.1-0.45 microm pore size can retain bacteria. In contrast to this assumption, we have regularly observed the passage of a significant fraction of natural freshwater bacterial communities through 0.45, 0.22, and 0.1 microm pore size filters. Flow cytometry and a regrowth assay were applied in the present study to quantify and cultivate filterable bacteria. Here we show for the first time a systematic quantification of their filterability, especially their ability to pass through 0.1 microm pore size filters. The filtered bacteria were subsequently able to grow on natural assimilable organic carbon (AOC) with specific growth rates up to 0.47 h(-1). We were able to enrich bacteria communities that pass preferentially through all three pore size filters at significantly increased percentages using successive filtration-regrowth cycles. In all instances, the dominant microbial populations comprised slender spirillum-shaped Hylemonella gracilis strains, suggesting shape-dependent selection during the filtration process. This quantification of the omnipresence of microfilterable bacterial in natural freshwater and their regrowth characteristics demand a change in the sterile filtration practice used in industrial and engineering applications as well as scientific research.

Wang Y, Hammes F, Boon N, Egli T (2007) Quantification of the filterability of freshwater bacteria through 0.45, 0.22, and 0.1 mu m pore size filters and shape-dependent enrichment of filterable bacterial communities. Environmental Science & Technology 41 :7080-7086

<Go to ISI> ://000250110800035

Micro-filtration is a standard process for sterilization in scientific research, medical, and industrial applications, and to remove particles in drinking water or wastewater treatment. It is generally assumed, and confirmed by quantifying filtration efficiency by plating, that filters with a 0.1 - 0.45 mu m pore size can retain bacteria. In contrast to this assumption, we have regularly observed the passage of a significant fraction of natural freshwater bacterial communities through 0.45, 0.22, and 0.1 mu m pore size filters. Flow cytometry and a regrowth assay were applied in the present study to quantify and cultivate filterable bacteria. Here we show for the first time a systematic quantification of their filterability, especially their ability to pass through 0.1,urn pore size filters. The filtered bacteria were subsequently able to grow on natural assimilable organic carbon (AOC) with specific growth rates up to 0.47 h(-1). We were able to enrich bacteria communities that pass preferentially through all three pore size filters at significantly increased percentages using successive filtration - regrowth cycles. In all instances, the dominant microbial populations comprised slender spirillum-shaped Hylemonella gracilis strains, suggesting shape-dependent selection during the filtration process. This quantification of the omnipresence of microfilterable bacterial in natural freshwater and their regrowth characteristics demand a change in the sterile filtration practice used in industrial and engineering applications as well as scientific research.

Yu Y, Kong FX, Wang ML, Qian LL, Shi XL (2007) Determination of short-term copper toxicity in a multispecies microalgal population using flow cytometry. Ecotoxicology and Environmental Safety 66 :49-56

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This study was conducted to determine the role of algal-algal interactions in a multispecies microalgal population on their sensitivities to copper based on an enzyme inhibition assay using flow cytometric measures. Autofluorescence (chlorophyll a and phycocyanin) was used to identify species and count algal signals. The effect of multispecies population on copper toxicity of Microcystis aeruginousa was detected (1) at the same initial cell density, (2) at the same surface area, and (3) in the presence and absence of Chlorella pyrenoidosa and Scenedesmus obliquus. As copper concentrations increased, esterase activity of M. aeruginosa changed in a concentration-dependent manner. The 24 h EC50 value of M. aeruginosa in the multispecies population was significantly (P < 0.05) higher than those in the single-species population. Compared with S. obliquus, the effect of C pyrenoidosa on M. aeruginosa was more marked (the 24 h EC50 value of copper on fluorescin diacetate fluorescence of M. aeruginosa was 11 mu g/L). At 48 h copper exposure (6 mu g/L) analysis of intracellular reactive oxygen species levels also showed similar algal-algal interactions in multispecies microalgal populations. The pigment assay suggested that these algal-algal interactions occurred only at low concentrations (< 13 mu g/L, 24 and 48 h copper exposure). This study demonstrates the importance of using multispecies populations to estimate metal toxicity in natural waters. (c) 2005 Elsevier Inc. All rights reserved.

Zhang L, Bao Z, Cheng J, Li H, Huang X, Wang S, Zhang C, Hu J (2007) Fosmid library construction and initial analysis of end sequences in Zhikong scallop (Chlamys farreri). Mar Biotechnol (NY) 9 :606-612


Zhikong scallop (Chlamys farreri Jones et Preston, 1904) is one of the most commercially important bivalves in China, but research on its genome is underdeveloped. In this study, we constructed the first Zhikong scallop fosmid library, and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between two and eight positive clones, and none of those tested was absent from the library. End-sequencing of 480 individual clones generated 828 sequences after trimming, with an average sequence length of 624 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 213 (25.72%) and 44 (5.31%) significant hits (E < e(-5)), respectively. Repetitive sequences analysis resulted in 375 repeats, accounting for 15.84% of total length, which were composed of interspersed repetitive sequences, tandem repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of the Zhikong scallop genome.

Zhang M, Kong FX, Shi XL, Xing P, Tan X (2007) Differences in responses to darkness between Microcystis aeruginosa and Chlorella pyrenoidosa. Journal of Freshwater Ecology 22 :93-99

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The effect of darkness on the cyanobacterium Microcystis aeruginosa and the green alga Chlorella pyrenoidosa were observed. Cell growth, metabolic activity, chlorophyll a fluorescence, cell size, and maximum quantum yield (F-v : F-m) were determined. M aeruginosa and C pyrenoidosa both maintained their initial cell abundances while in the dark, but they showed distinctly different physiological responses to darkness. During the dark period metabolic activity decreased slightly, cell size and F-v : F-m decreased significantly, and chlorophyll a increased markedly in M aeruginosa cells. However, metabolic activity, cell size, and F-v : F-m decreased dramatically, and chlorophyll a decreased trivially in C. pyrenoidosa cells. After re-illumination, metabolic activity, cell size, and F-v : F-m, of M. aeruginosa and metabolic activity, chlorophyll a, and F-v : F-m of C pyrenoidosa recovered significantly. No recovery was found in either chlorophyll a of M aeruginosa or cell size of C pyrenoidosa. Compared with C. pyrenoidosa, M aeruginosa is more capable of withstanding the dark circumstances, which are unfavorable to the growth of alga but usually exists in water and sediment of eutrophic lakes. Its greater tolerance of darkness may be one of the reasons that M aeruginosa is commonly the dominant species in eutrophic takes.

Zhang M, Kong FX, Xing P, Tan X (2007) Effects of interspecific interactions between Microcystis aeruginosa and Chlorella pyrenoidosa on their growth and physiology. International Review of Hydrobiology 92 :281-290

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Interactions between Microcystis aeruginosa and Chlorella pyrenoidosa were analyzed by flow cytometry and by phytoplankton pulse-amplitude-modulated fluorimetry (Phyto-PAM) in joint cultures as well as in cultures separated by dialysis membranes. Results showed that the growth of C. pyrenoidosa was greater than that of M. aeruginosa, and that the growth of M. aeruginosa but not the growth of C. pyrenoidosa was significantly inhibited by the interactions between M. aeruginosa and C. pyrenoidosa. Culture filtrates of these two algae showed no apparent effects on the growth of the competing species. For M. aeruginosa, decreases in esterase activity, chlorophyll a fluorescence, and maximum quantum yield were observed in joint cultures, indicating that the metabolic activity and photosynthetic capacity of M. aeruginosa were suppressed. Light limitation from the shading effect of C. pyrenoidosa may be the main reason for such inhibition. For C. pyrenoidosa, esterase activity was suppressed in membrane-separated and joint cultures, suggesting that C. pyrenoidosa was probably affected by allelopathic substances secreted by M. aeruginosa. However, no significant difference was observed in the chlorophyll a fluorescence and maximum quantum yield of C. pyrenoidosa in the two cultures. In addition, interspecific interactions induced a reduction in size in both M. aeruginosa and C. pyrenoidosa, which may contribute to the development of C. pyrenoidosa dominance in the present study.

Zhang R, Weinbauer MG, Qian PY (2007) Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters. Environmental Microbiology 9 :3008-3018

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To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.

Zubkov M, Burkill PH, Topping JN (2007) Flow cytometric enumeration of DNA-stained oceanic planktonic protists. Journal of Plankton Research 29 :79-86

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The aim of this study was to test the practicality of enumerating fixed, DNA-stained heterotrophic protists (H) and phototrophic protists (P) in contrasting regions of the Atlantic Ocean. Oceanic protists were enumerated using a standard flow cytometer (FACSort, BD) at an enhanced flow rate of up to 1.0 mL min(-1) to increase numbers of counted cells. The enumeration error of protists decreased hyperbolically from 30-40 to < 5% corresponding to the number (< 100 to > 2000) of enumerated cells. H and P were discriminated using the extra red chlorophyll-derived plastidic fluorescence of the latter. The relationship between counts of stained and unstained fixed and unfixed P was statistically close to 1:1, confirming the accuracy of stained protist counting by flow cytometry and adequate discrimination of P from H cells. The estimated average abundance of H in the surface mixed layer of the southern and northern oligotrophic gyres was remarkably similar, with 400 +/- 140 and 450 +/- 60 cells mL(-1), respectively, adding further evidence to the suggestion that these regions are in steady state. In agreement with earlier studies in more productive aquatic environments, a significant correlation (correlation coefficient 0.84, P < 0.0001) was found between the H and the total bacterioplankton numbers, with an average ratio of similar to 1300 prokaryotes to 1 H cell, suggesting a relatively constant trophic interaction between these two groups. This study demonstrates that flow cytometric enumeration of protists is similar to 100 times faster compared with microscopy and, thus, represents a major improvement for quantifying protists in ocean waters, including oligotrophic gyres.

Zubkov MV, Holland RJ, Burkill PH, Croudace IW, Warwick PE (2007) Microbial abundance, activity and iron uptake in vicinity of the Crozet Isles in November 2004-January 2005. Deep-Sea Research Part Ii-Topical Studies in Oceanography 54 :2126-2137

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Iron leached from volcanic islands was hypothesised to naturally fertilise the high-nutrient low-chlorophyll (HNLC) waters of the Antarctic Circum Polar Current and to cause recurrent phytoplankton blooms or high-chlorophyll (HQ areas in the wake of the Crozet Islands. As part of CROZEX, the effect of Fe-fertilisation on microbial community was examined by comparing microbial standing stocks and microbial turnover rates of dissolved organic molecules and iron in the HNLC and HC waters in the vicinity of the Crozet Isles. Bacterioplankton and ultraplanktonic algae were enumerated by flow cytometry. Microbial turnover and ambient concentrations of amino acids and glucose in surface waters were bioassayed using an isotopic dilution technique. Microbial uptake of iron was estimated using a carrier-free Fe-55 tracer approach. The data set generated did not reveal statistically significant seasonal changes above the observed high spatial variability in the studied area. Statistically significant higher biomass (1.5 times) of heterotrophic bacterioplankton (HB) as well as higher microbial turnover of organic molecules (10 times) were observed in the HC waters relative to the HNLC waters, while relative iron uptake was nearly eight times lower in the HC waters. However, the difference in HB standing stocks in the 100-200 m water layer between the two compared water types was statistically insignificant. Hence, the HC surface waters in austral summer showed higher microbial activity with decreased iron dependency relative to the HNLC waters of the Southern Ocean, in agreement with higher productivity of the waters to the north of the Crozet Islands. (C) 2007 Elsevier Ltd. All rights reserved.

Zubkov MV, Mary I, Woodward EMS, Warwick PE, Fuchs BM, Scanlan DJ, Burkill PH (2007) Microbial control of phosphate in the nutrient-depleted North Atlantic subtropical gyre. Environmental Microbiology 9 :2079-2089

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Little is known about the dynamics of dissolved phosphate in oligotrophic areas of the world’s oceans, where concentrations are typically in the nanomolar range. Here, we have budgeted phosphate uptake by the dominant microbial groups in order to assess the effect of the microbial control of this depleted nutrient in the North Atlantic gyre. Low concentrations (2.2 +/- 1.2 nM) and rapid microbial uptake (2.1 +/- 2.4 nM day(-1)) of bioavailable phosphate were repeatedly determined in surface waters of the North Atlantic oligotrophic gyre during spring and autumn research cruises, using a radiotracer dilution bioassay technique. Upper estimates of the concentration of bioavailable phosphate were 7-55% of the dissolved mineral phosphate suggesting that a considerable part of the chemically measured nanomolar phosphate was in a form unavailable for direct microbial uptake. A 1:1 relationship (r(2) = 0.96, P < 0.0001) was observed between the bioavailable total phosphate uptake and the phosphate uptake of all the flow sorted bacterioplankton cells, demonstrating that bacterioplankton were the main consumers of phosphate. Within the bacterioplankton a group of heterotrophic bacteria and Prochlorococcus phototrophic cyanobacteria, were the two major competing groups for bioavailable phosphate. These heterotrophic bacteria had low nucleic acid content and 60% of them comprised of SAR11 clade cells based on the results of fluorescence in situ hybridization. Each of the two competing bacterial groups was responsible for an average of 45% of the phosphate uptake, while Synechococcus cyanobacteria (7%) and picoplanktonic algae (0.3%) played minor roles in direct phosphate uptake. We have demonstrated that phosphate uptake in the oligotrophic gyre is rapid and dominated by two bacterial groups rather than by algae.