mardi 21 avril 2009
par   G. Grégori

Agawin NSR, Hale MS, Rivkin RB, Matthews P, Li WKW (2006) Microbial response to a mesoscale iron enrichment in the NE Subarctic Pacific : Bacterial community composition. Deep-Sea Research Part Ii-Topical Studies in Oceanography 53 :2248-2267

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Changes in microbial community composition were determined during the subarctic ecosystem response to iron enrichment study (SERIES), a mesoscale Fe enrichment conducted in a high-nutrient low-chlorophyll (HNLC) region of the Northeast Subarctic Pacific, in July 2002. Phylogenetic composition using fluorescence in situ hybridization (FISH), relative DNA content using flow cytometry (FCM), and cellular morphometrics (shape and volume) of heterotrophic bacteria were used to characterize community composition from samples collected within and below the mixed layer, inside and outside the Fe-patch. The proportion of total cells detected as members of the Cytophaga-Flavobacterium cluster increased in a log-linear manner from 16 (+/- 1.0)% to 47 (+/- 1.9)% in samples within the mixed layer, inside the Fe-enriched patch, while outside the patch, the proportion remained <= 21 (+/- 2.2)%. Temporal changes in the proportion of cells in the mixed layer with high DNA content (% HDNA) were significantly different inside and outside the Fe-enriched patch, where inside the patch % HDNA increased 2-fold after a week, reaching 93% towards the end of the observation period. Coupling in situ observations with the results of manipulation experiments allowed us to determine the relative contributions of bottom-up (nutrient limitation) and top-down (grazing) processes on heterotrophic bacterial abundance and community composition. Shifts in heterotrophic bacterial community composition inside the Fe-enriched patch were mainly controlled by top-down processes and moderately controlled by bottom-up controls (organic substrate limitation). (c) 2006 Elsevier Ltd. All rights reserved.

Akselband Y, Cabral C, Castor TP, Chikarmane HM, McGrath P (2006) Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting. Journal of Experimental Marine Biology and Ecology 329 :196-205

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Encapsulation of cells in agarose gel microdrops (GMDs) combined with fluorescence-activated cell sorting (FACS) has been used previously to analyze and recover specific mammalian, bacterial, and yeast cell populations. Recently, we have developed a method to enrich mixed bacterial populations for slow-growing microorganisms using the GMD Growth Assay combined with fluorochrome staining and flow cytometry. Here, we demonstrate the feasibility of using this experimental approach to detect clonogenic growth of individual bacteria within GMDs in less than 3 h and to separate subpopulations based on differential growth rates. We show that after sorting, organisms remain viable and can be propagated in culture for further analysis. (c) 2005 Elsevier B.V. All rights reserved.

Allam B, Ford SE (2006) Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species : I. Haemocyte changes following in vitro challenge. Fish Shellfish Immunol 20 :374-383


In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species : the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.

Alonso-Saez L, Gasol JM, Lefort T, Hofer J, Sommaruga R (2006) Effect of natural sunlight on bacterial activity and differential sensitivity of natural bacterioplankton groups in northwestern Mediterranean coastal waters. Appl Environ Microbiol 72 :5806-5813


We studied the effects of natural sunlight on heterotrophic marine bacterioplankton in short-term experiments. We used a single-cell level approach involving flow cytometry combined with physiological probes and microautoradiography to determine sunlight effects on the activity and integrity of the cells. After 4 h of sunlight exposure, most bacterial cells maintained membrane integrity and viability as assessed by the simultaneous staining with propidium iodide and SYBR green I. In contrast, a significant inhibition of heterotrophic bacterial activity was detected, measured by 5-cyano-2,3 ditolyl tetrazolium chloride reduction and leucine incorporation. We applied microautoradiography combined with catalyzed reporter deposition-fluorescence in situ hybridization to test the sensitivity of the different bacterial groups naturally occurring in the Northwestern Mediterranean to sunlight. Members of the Gammaproteobacteria and Bacteroidetes groups appeared to be highly resistant to solar radiation, with small changes in activity after exposure. On the contrary, Alphaproteobacteria bacteria were more sensitive to radiation as measured by the cell-specific incorporation of labeled amino acids, leucine, and ATP. Within Alphaproteobacteria, bacteria belonging to the Roseobacter group showed higher resistance than members of the SAR11 cluster. The activity of Roseobacter was stimulated by exposure to photosynthetic available radiation compared to the dark treatment. Our results suggest that UV radiation can significantly affect the in situ single-cell activity of bacterioplankton and that naturally dominating phylogenetic bacterial groups have different sensitivity to natural levels of incident solar radiation.

Auffret M, Rousseau S, Boutet I, Tanguy A, Baron J, Moraga D, Duchemin M (2006) A multiparametric approach for monitoring immunotoxic responses in mussels from contaminated sites in Western Mediterranea. Ecotoxicology and Environmental Safety 63 :393-405

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As a part of the multidisciplinary program Biological Effects of Environmental Pollution in Marine Coastal Ecosystems of the European Commission, this study aimed to validate immunological alterations as biomarkers of exposure to chemical contamination in polluted areas of Western Mediterranea. The status of the immune system has been assessed in mussels (Mytilus galloprovincialis) by measuring several immunopathological and immunocompetence parameters. Alterations of total hemocyte counts, lysosomal stability, and phagocytosis were among the most reliable effects observed in polluted sites and suggested immunosuppressive conditions in contaminated mussels. An immunotoxicological index was calculated from the set of individual data. By providing a single value per sampling station to score immunological alterations in mussels, this novel approach allowed recognition of a gradient of perturbation correlated to pollution intensity in two of the three sites monitored. Processing a set of biological parameters by this method was found to increase the ecotoxicological relevance of such multiparametric studies for the assessement of chemical contamination in coastal waters. (c) 2005 Elsevier Inc. All rights reserved.

Barnaba F, Fiorani L, Palucci A, Tarasov P (2006) First characterization of marine particles by laser scanning flow cytometry. Journal of Quantitative Spectroscopy & Radiative Transfer 102 :11-17

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A new laser scanning flow cytometer (CLASS) has been developed at ENEA to characterize single marine particles. The first setup of the instrument allowing the detection of the particle light scattering over a wide angular range is presented. A test of CLASS employing polystyrene microspheres is described. Eventually, the first measurements of biological particles (Penicillium Italicum conidia) and marine phytoplankton (Synechocystis cells) are reported. (c) 2006 Elsevier Ltd. All rights reserved.

Beineke A, Siebert U, Baumgartner W (2006) The immune system of marine mammals. Part 2 : Morphology, immunophenotyping and pathology of lymphoid organs. Tieraerztliche Praxis Ausgabe Kleintiere Heimtiere 34 :201-207

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The majority of primary and secondary lymphoid organs of whales and dolphins are homologous to those organs of terrestrial mammals. However, some unique anatomical structures, such as the complex lymp-hoepithelial laryngeal glands in cetaceans represent an adaptation to the marine environment. Additionally, physiologic changes, such as age-related thymic atrophy and cystic degeneration of the "anal tonsil" of whales have to be considered in the interpretation of pathologic lesions in these lymphoid structures. Specific leukocyte-markers enable the detailed characterization of cellular responses during immunological and inflammatory processes. Using immunological methods, such as flow cytometry and immunohistochemistry, different T and B cell subsets as well as antigen-presenting cells can be detected in whales and dolphins. However, only a few reports mention the usefulness of cross-reacting or species-specific antibodies in these animals. Pathological lesions in lymphoid organs can be observed during several infectious and neoplastic diseases in marine mammals. Regarding this, systemic morbillivirus infections lead to devastating mass mortalities associated with severe lymphoid depletions in cetaceans and pinnipeds worldwide. Furthermore, chronic diseases and starvation are associated with a loss of functional lymphoid cells and decreased resistance against opportunistic infections. Additionally, there is growing evidence for the immunotoxic effect of different environmental contaminants and their immunosuppressive potentials in whales, dolphins and seals.

Berney M, Weilenmann HU, Egli T (2006) Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS). Microbiology 152 :1719-1729


The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely : efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol ; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose ; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the ’agony’ of E. coli when it is stressed with sunlight.

Bigas M, Durfort M, Poquet M (2006) Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury. Biometals 19 :659-673

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Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.

Binet M, Stauber J, Franklin N, Adams M (2006) Applications of flow cytometry to marine ecotoxicology and ballast water studies. Cytometry Part A 69A :403-404

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Binet MT, Stauber JL (2006) Rapid flow cytometric method for the assessment of toxic dinoflagellate cyst viability. Mar Environ Res 62 :247-260


The inadvertent transfer and dispersal of non-indigenous marine species via shipping ballast water is of increasing environmental concern. Despite a major global effort to develop new ballast water treatment technologies, their acceptance has been hampered by the lack of suitable indicator species for assessing treatment effectiveness. Resistant dinoflagellate cysts are one proposed test organism, however their use has been limited due to difficulties in assessing their viability after treatment. The paper describes the development of a rapid method to determine the viability of cysts of the dinoflagellate Alexandrium catenella using staining with SYTOX Green and flow-cytometric analysis. The viability of A. catenella cysts was inversely proportional to their ability to take up the stain. There was excellent agreement between cysts measured as viable/non-viable using flow cytometry and cyst viability determined in standard long-term germination tests. Advantages of the flow-cytometric method include high test precision and rapid testing times of < 2 days, compared to > 4 weeks using existing germination methods.

Bouilly K, Gagnaire B, Bonnard M, Thomas-Guyon H, Renault T, Miramand P, Lapegue S (2006) Effects of cadmium on aneuploidy and hemocyte parameters in the Pacific oyster, Crassostrea gigas. Aquat Toxicol 78 :149-156


Pacific oysters, Crassostrea gigas, are commonly reared in estuaries where they are exposed to anthropogenic pollution. Much research has been made on the toxicity of cadmium to aquatic organisms because the compound recurrently contaminates their environment. Our study examined the influence of cadmium on aneuploidy level (lowered chromosome number in a percentage of somatic cells) and hemocyte parameters in C. gigas at different stages of life. Adults and juveniles were exposed to two different concentrations of cadmium. The first concentration applied was equivalent to a peak value found in Marennes-Oleron bay (Charente-Maritime, France ; 50 ngL(-1)) and the second was 10 times higher (500 ngL(-1)). Exposure to 50 ngL(-1) cadmium caused a significant decrease in the survival time of C. gigas, but exposure to 500 ngL(-1) surprisingly affected the survival time positively. Significant differences in aneuploidy level were observed between the cadmium treatments and the control in adults but not in juveniles or the offspring of the adult groups. The effects of cadmium on hemocyte parameters were analyzed by flow cytometry. Several hemocyte parameters increased significantly after 21 days of cadmium exposure and subsequently decreased. Phenoloxidase-like activity, evaluated by spectrophotometry, varied over the time of the experiment and increased after 66 days of contact with 500 ngL(-1) cadmium. Taken together, cadmium at environmentally relevant concentrations seems to have only moderate effects on aneuploidy and hemocyte parameters.

Bouman HA, Ulloa O, Scanlan DJ, Zwirglmaier K, Li WK, Platt T, Stuart V, Barlow R, Leth O, Clementson L, Lutz V, Fukasawa M, Watanabe S, Sathyendranath S (2006) Oceanographic basis of the global surface distribution of Prochlorococcus ecotypes. Science 312 :918-921


By using data collected during a continuous circumnavigation of the Southern Hemisphere, we observed clear patterns in the population-genetic structure of Prochlorococcus, the most abundant photosynthetic organism on Earth, between and within the three Southern Subtropical Gyres. The same mechanisms that were previously invoked to account for the vertical distribution of ecotypes at local scales accounted for the global (horizontal) patterns we observed. Basin-scale and seasonal variations in the structure and strength of vertical stratification provide a basis for understanding large-scale horizontal distribution in genetic and physiological traits of Prochlorococcus, and perhaps of marine microbial communities in general.

Bouvy M, Pagano M, M’Boup M, Got P, Troussellier M (2006) Functional structure of microbial food web in the Senegal River Estuary (West Africa) : impact of metazooplankton. Journal of Plankton Research 28 :195-207

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We studied the impact of various metazooplanktonic predators (two calanoid copepods and barnacle larvae) on microbial plankton isolated from the Senegal River Estuary. Experiments performed in microcosms were based on size-class fractionation (3, 12, 20 and 60 mu m size classes) of the microbial community in order to obtain different assemblages, including bacteria, heterotrophic nanoflagellates (HNF), ciliates, pico- and nanophytoplankton. Removal of bacterial predators > 3 mu m increased the net growth rate of heterotrophic bacteria from 0.437 day(-1) in the presence of all predators (fraction < 60 mu m) to 0.682 day(-1) (fraction < 3 mu m). Removal of protozoa > 12 mu m caused an increased net growth rate of flagellates from 0.102 day(-1) (fraction < 60 mu m) to 0.332 day(-1) (fraction < 12 mu m). The same results were also observed on protozoa dynamics in the presence of Temora stylifera (0.498 day(-1)), suggesting a trophic control of the protozoa > 12 mu m by this copepod, allowing an increase in HNF growth rates through indirect effect. The highest ingestion rate over 60 h of experiment was recorded for the copepod Temora (61.1 mu m(3) ngC(-1) h(-1)) and the lowest value for barnacle larvae (25.6 mu m(3) ngC(-1) h(-1)). An intermediate value (47.5 mu m(3) ngC(-1) h(-1)) was recorded for the copepod Acartia clausi. All these ingestion rates were mainly channeled in the size classes between < 3 and 15-18 mu m equivalent spherical diameter (ESD) (6 and 2435 mu m(3), respectively), with the highest values noted between < 3 and 6-9 mu m classes. In the presence of Temora, the cascading effects can explain both the decreases in mean volume and in growth rate for bacteria, due to the filtration of ciliates by the zooplanktonic predator, allowing the development of the HNF. The presence of a large size class of bacteria (among the five classes identified by flow cytometry) and a high abundance of HNF suggest an efficient heterotrophic pathway within the microbial loop of this sub-saharan estuary.

Brunet C, Casotti R, Vantrepotte V, Corato F, Conversano F (2006) Picophytoplankton diversity and photoacclimation in the Strait of Sicily (Mediterranean Sea) in summer. I. Mesoscale variations. Aquatic Microbial Ecology 44 :127-141

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Phytoplankton dynamics were investigated at the mesoscale in the northern part of the Strait of Sicily in July-August 1997 on fractionated samples (< 3 and > 3 pm) using HPLC pigment analysis and flow cytometry. Distribution, diversity and photoacclimation varied within the different water masses and features present at the time of sampling, including a surface filament of deep, cold water. Picophytoplankton (< 3 mu m) accounted for 80% of total chlorophyll on average, and was numerically dominated by cyanobacteria of the genus Prochlorococcus, with an average concentration of 5.2 x 10(4) cells ml(-1). The biomass and pigment diversity of picophytoplankton was higher in the deep chlorophyll maximum (DCM) and was related to hydrological and biological features, whereas larger phytoplankton (> 3 mu m) appeared to respond to different cues. Chlorophyll pigment content per cell of Synechococcus spp., Prochlorococcus spp. or picoeukaryotes was estimated by coupling pigment data with flow cytometric counts. In Prochlorococcus spp., we found an average of 0.44 and 1.56 fg divinyl-chlorophyll a (dvchl a) cell(-1) in surface and DCM layers, respectively. In contrast, chl a content in the picoeukaryote group ranged between 17 and 168 fg chl a cell(-1), depending upon depth and water mass, which suggested strong photoacclimation and photoadaptation with depth. The relative contribution of each eukaryote pigment to one size class or the other changed through the water column, and reflected size segregation within single taxonomic groups.

Caroppo C, Stabili L, Aresta M, Corinaldesi C, Danovaro R (2006) Impact of heavy metals and PCBs on marine picoplankton. Environmental Toxicology 21 :541-551

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Synergistic/antagonistic effects of multiple contaminants in marine environments are almost completely unexplored. In the present study, we investigated the effects of heavy metals (Zn and Pb) and PCBs on picoplankton abundance, biomass, cell size distribution, and bacterial C production. Natural picoplankton assemblages were exposed to heavy metals (Zn or Pb), organic contaminants (PCBs, Aroclor 1260), and to a mixture of different contaminants. The results of the present study indicate that Zn addition stimulated heterotrophic growth, whereas Pb has a negative impact on heterotrophic picoplankton, particularly significant in the first 24 h. Heavy metals had no effects on the autotrophic component. The addition of Aroclor 1260 had a significant impact on abundance, biomass, and cell size of autotrophic and heterotrophic picoplankton, and reduced significantly bacterial secondary production. Three weeks after PCB treatment, heterotrophic bacteria displayed a clear resilience, both in terms of abundance and biomass, reaching values comparable to those of the controls, but not in terms of bacterial C production. Our results indicate that picoplankton can be sensitive indicators of impact determined by heavy metals and PCBs in coastal marine systems. (c) 2006 Wiley Periodicals, Inc.

Carvalho WF, Graneli E (2006) Acidotropic probes and flow cytometry : a powerful combination for detecting phagotrophy in mixotrophic and heterotrophic protists. Aquatic Microbial Ecology 44 :85-96

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Studies with phagotrophic organisms are hampered by a series of methodological constraints. To overcome problems related to the detection and enumeration of mixotrophic and heterotrophic cells containing food vacuoles, we combined flow cytometry and an acidotropic blue probe as an alternative method. Flow cytometry allows the analysis of thousands of cells per minute with high sensitivity to the autofluorescence of different groups of cells and to probe fluorescence. The method was first tested in a grazing experiment where the heterotrophic dinoflagellate Oxyrrhis marina fed on Rhodomonas salina. The maximum ingestion rate of O. marina was 1.7 prey ind.(-1) h(-1), and the frequency of cells with R. salina in the food vacuoles increased from 0 to 2.4 +/- 0.5 x 10(3) cells ml(-1) within 6 h. The blue probe stained 100% of O. marina cells that had R. salina in the food vacuoles. The acidotropic blue probe was also effective in staining food vacuoles in the mixotrophic dinoflagellate Dinophysis norvegica. We observed that 75% of the D. norvegica population in the aphotic zone possessed food vacuoles. Overall, in cells without food vacuoles, blue fluorescence was as low as in cells that were kept probe free. Blue fluorescence in O. marina cells with food vacuoles was 6-fold higher than in those without food vacuoles (20 +/- 4 and 3 +/- 0 relative blue fluorescence cell(-1), respectively), while in D. norvegica cells were 4.5-fold brighter than the ones without food vacuoles (291 +/- 155 and 64 +/- 23 relative blue fluorescence cell(-1), respectively). The use of acidotropic probes can prevent fixation artifacts such as regurgitation of food vacuoles and changes in the cellular characteristics. The combination of flow cytometry and an acidotropic probe proved to be an efficient tool in detecting phagotrophy in mixotrophic and heterotrophic marine phytoplankton species.

Cottrell MT, Mannino A, Kirchman DL (2006) Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre. Appl Environ Microbiol 72 :557-564


The abundance of aerobic anoxygenic phototrophic (AAP) bacteria, cyanobacteria, and heterotrophs was examined in the Mid-Atlantic Bight and the central North Pacific Gyre using infrared fluorescence microscopy coupled with image analysis and flow cytometry. AAP bacteria comprised 5% to 16% of total prokaryotes in the Atlantic Ocean but only 5% or less in the Pacific Ocean. In the Atlantic, AAP bacterial abundance was as much as 2-fold higher than that of Prochlorococcus spp. and 10-fold higher than that of Synechococcus spp. In contrast, Prochlorococcus spp. outnumbered AAP bacteria 5- to 50-fold in the Pacific. In both oceans, subsurface abundance maxima occurred within the photic zone, and AAP bacteria were least abundant below the 1% light depth. The abundance of AAP bacteria rivaled some groups of strictly heterotrophic bacteria and was often higher than the abundance of known AAP bacterial genera (Erythrobacter and Roseobacter spp.). Concentrations of bacteriochlorophyll a (BChl a) were low ( approximately 1%) compared to those of chlorophyll a in the North Atlantic. Although the BChl a content of AAP bacteria per cell was typically 20- to 250-fold lower than the divinyl-chlorophyll a content of Prochlorococcus, the pigment content of AAP bacteria approached that of Prochlorococcus in shelf break water. Our results suggest that AAP bacteria can be quite abundant in some oceanic regimes and that their distribution in the water column is consistent with phototrophy.

Countway PD, Caron DA (2006) Abundance and distribution of Ostreococcus sp. in the San Pedro Channel, California, as revealed by quantitative PCR. Appl Environ Microbiol 72 :2496-2506


Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 mum) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 x 10(3) cells ml(-1), with a maximum of 8.2 x 10(4) cells ml(-1). Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 x 10(5) cells ml(-1). The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was approximately 70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was approximately 30 mg C m(-2). Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.

Cox EF, Ribes M, Kinzie RA (2006) Temporal and spatial scaling of planktonic responses to nutrient inputs into a subtropical embayment. Marine Ecology-Progress Series 324 :19-35

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We carried out a study of the spatial and temporal effects of land-derived material on water column nutrients and plankton dynamics in a subtropical estuary. The study had 2 parts : (1) a 3 yr synoptic monitoring program, and (2) a shorter 1.5 yr study during the second half of the program, which focused on individual pulses driven by discrete rainfall events. Although we found spatial differences in some water column parameters within Kane’ohe Bay and an adjacent oceanic site, inorganic nutrient levels were generally comparable in the Bay and offshore. One difference was that Prochlorococcus spp. numerically dominated the plankton at the oceanic site whereas Synechococcus spp. dominated at all Bay sites. The switch in dominance appears to be due to light characteristics and dissolved organic nitrogen (DON), but not dissolved inorganic nutrient availability. There were no annual cycles in water column parameters within the Bay ; however, a comparison of dry and wet seasons did show some differences. Planktonic cell abundance was in general lower during the wet season, with the exception of opportunistic diatoms that were more abundant during the wet season. A drought during the study period may have influenced our results. Pulses were characterized by an elevation in inorganic nutrient concentrations in the Bay close to the stream mouth. The general response was an increase in abundance of microphytoplankton and chl a after a 3 to 6 d lag following the nutrient increase. Picophytoplankton showed an increase in fluorescence per cell after a 12 to 24 h lag, probably related to a decrease in irradiance associated with turbidity in runoff. The Bay can act as source of dissolved inorganic nutrients and plankton for oceanic waters ; however, planktonic populations in the Bay are primarily autochthonous and do not represent an oceanic source of nutrients for plankton consumers within Kane’ohe Bay.

Cresswell T, Richards JP, Glegg GA, Readman JW (2006) The impact of legislation on the usage and environmental concentrations of Irgarol 1051 in UK coastal waters. Marine Pollution Bulletin 52 :1169-1175

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In 2001, legislative measures were introduced in the UK to restrict usage of antifouling agents in small (< 25 m) vessel paints to dichlofluanid, zinc pyrithione and zineb. This removed the previously popular booster biocides diuron and irgarol 10510 from the market. To investigate the impact of this legislation, water samples were taken from locations where previous biocide levels were well documented. Results from analyses demonstrate a clear reduction in water concentrations of Irgarol 1051 (between 10% and 55% of that found during pre-restriction studies), indicating that legislation appears to have been effective. Although other booster biocides were screened for (chlorothalonil, dichlofluanid and Sea-Nine 2110), they were below the limits of detection (< 1 ng/l) in all samples. A survey of chandlers and discussions with legislative authorities supports these results and concurs the removal of Irgarol 1051 based paints from the market using simple regulations at a manufacturer level with little regulation at a retailer level. (c) 2006 Elsevier Ltd. All rights reserved.

da Silva JAT, Chan MT, Sanjaya, Chai ML, Tanaka M (2006) Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction, plantlet formation, and their subsequent cytogenetic stability analysis. Scientia Horticulturae 109 :368-378

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Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ’Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg 1(-1) each, respectively, both within 45-60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite ; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis ; coconut water, when supplied at 10-20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity ; Fe-EDTA (I mg F 1) and activated charcoal (I g F 1) stimulated total fresh weight and PLB formation in the presence of PGRs ; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo-inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 mu mol m(-2) s(-1) ; at 15 mu mol m(-2) s(-1) shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment. (c) 2006 Elsevier B.V. All rights reserved.

Davy SK, Burchett SG, Dale AL, Davies P, Davy JE, Muncke C, Hoegh-Guldberg O, Wilson WH (2006) Viruses : agents of coral disease ? Dis Aquat Organ 69 :101-110


The potential role of viruses in coral disease has only recently begun to receive attention. Here we describe our attempts to determine whether viruses are present in thermally stressed corals Pavona danai, Acropora formosa and Stylophora pistillata and zoanthids Zoanthus sp., and their zooxanthellae. Heat-shocked P. danai, A. formosa and Zoanthus sp. all produced numerous virus-like particles (VLPs) that were evident in the animal tissue, zooxanthellae and the surrounding seawater ; VLPs were also seen around heat-shocked freshly isolated zooxanthellae (FIZ) from P. danai and S. pistillata. The most commonly seen VLPs were tail-less, hexagonal and about 40 to 50 nm in diameter, though a diverse range of other VLP morphotypes (e.g. rounded, rod-shaped, droplet-shaped, filamentous) were also present around corals. When VLPs around heat-shocked FIZ from S. pistillata were added to non-stressed FIZ from this coral, they resulted in cell lysis, suggesting that an infectious agent was present ; however, analysis with transmission electron microscopy provided no clear evidence of viral infection. The release of diverse VLPs was again apparent when flow cytometry was used to enumerate release by heat-stressed A. formosa nubbins. Our data support the infection of reef corals by viruses, though we cannot yet determine the precise origin (i.e. coral, zooxanthellae and/or surface microbes) of the VLPs seen. Furthermore, genome sequence data are required to establish the presence of viruses unequivocally.

Diao Y, Chen L, Yang GX, Zhou MQ, Song YC, Hu ZL, Liu JY (2006) Nuclear DNA C-values in 12 species in nymphaeales. Caryologia 59 :25-30

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Nuclear DNA C-values are the basic data of species and used in a strikingly wide range of biological fields. Genome size of the most species belonging to the three families Nulembonaceae, Cabombaceae and Nymphaeaceae in Nymphaeales were not assessed, especially none of the species in Cabombaccae was reported so far. In present research, flow cytometry was used to assess the genome size of 12 species belonging to the three families in Nymphaeales and a standard squash technique to count the chromosome numbers of the tested materials. In the tested different species, 2C nuclear DNA contents ranged from 1.55 to 8.11 pg, which is more than fivefold variation. And chromosome numbers varied from 2n=16 to 2n=72. Differences between the species or among populations within a species were also recorded by statistical analysis. The tested species are all small genomes except two species of Victoria. The ploidies did not matched with their DNA contents very well in Nympbaea. Significant differences were found between the species of Nymphaea, between those of Victoria and among wild populations of Nelumbo nicifera ; while no obvious differences were found between the species of Nelumbo and among the cultivars of Euryale ferox as well as among those of Brasenia schreberi, respectively.

Diaz MR, Boekhout T, Theelen B, Bovers M, Cabanes FJ, Fell JW (2006) Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species. J Med Microbiol 55 :1197-1209


Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as ’microcodes’ for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.

Dong QX, Huang CJ, Henk MC, Tiersch TR (2006) Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas. Cell and Tissue Research 324 :335-345

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Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed ; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P < 0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.

Du B, Wang D (2006) C-values of seven marine mammal species determined by flow cytometry. Zoolog Sci 23 :1017-1020


C-values, which estimate genome size, have puzzled geneticists for years because they bear no relationship to organismal complexity. Though C-values have been estimated for thousands of species, considerably more data are required in order to better understanding genome evolution. This is particularly true for mammals, in which C-values are known for less than 8% of the total number of mammalian species. Among marine mammals, a C-value has been estimated only for the bottlenose dolphin (Tursiops truncatus). Thus examination of additional species of marine mammals is necessary for comparative purposes. It will enable a better understanding of marine mammal genome evolution, and it is also relevant to conservation, because larger genome size has been linked to increased likelihood of extinction in some plant and animal groups. Our study presents C-values of seven marine mammal species, including five cetacean species that are endangered to varying degrees. Similarly to the results for other groups, our results suggest that larger genome size in cetaceans is related to an increased likelihood of extinction.

Duhamel S, Jacquet S (2006) Flow cytometric analysis of bacteria- and virus-like particles in lake sediments. J Microbiol Methods 64 :316-332


Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (x1/20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or -22 degrees C. For FCM analysis, the samples were diluted in Tris-EDTA buffer (pH 8) and heated for 10 min at 75 degrees C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.

Evans SL, Anderson WT, Jochem FJ (2006) Spatial variability in Florida Bay particulate organic matter composition : combining flow cytometry with stable isotope analyses. Hydrobiologia 569 :151-165

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Long-term management plans for restoration of natural flow conditions through the Everglades increase the importance of understanding potential nutrient impacts of increased freshwater delivery on Florida Bay biogeochemistry. Planktonic communities respond quickly to changes in water quality, thus spatial variability in community composition and relationships to nutrient parameters must be understood in order to evaluate future downstream impacts of modifications to Everglades hydrology. Here we present initial results combining flow cytometry analyses of phytoplankton and bacterial populations (0.1-50 mu m size fraction) with measurements of delta(13) C and delta N-15 composition and dissolved inorganic nutrient concentrations to explore proxies for planktonic species assemblage compositions and nutrient cycling. Particulate organic material in the 0.1-50 mu m size fraction was collected from five stations in Northeastern and Western Florida Bay to characterize spatial variability in species assemblage and stable isotopic composition. A dense bloom of the picocyanobacterium, Synechococcus elongatus, was observed at Western Florida Bay sites. Smaller Synechococcus sp. were present at Northeast sites in much lower abundance. Bacteria and detrital particles were also more abundant at Western Florida Bay stations than in the northeast region. The highest abundance of detritus occurred at Trout Creek, which receives freshwater discharge from the Everglades through Taylor Slough. In terms of nutrient availability and stable isotopic values, the S. elongatus population in the Western bay corresponded to low DIN (0.5 mu M NH4+ ; 0.2 mu M NO3-) concentrations and depleted delta N-15 signatures ranging from +0.3 to +0.8 parts per thousand, suggesting that the bloom supported high productivity levels through N-2-fixation. delta N-15 values from the Northeast bay were more enriched (+2.0 to + 3.0 parts per thousand), characteristic of N-recycling. delta C-13 values were similar for all marine Florida Bay stations, ranging from -17.6 to -14.47 parts per thousand, however were more depleted at the mangrove ecotone station (-25.5 to -22.3 parts per thousand). The difference in the isotopic values reflects differences in carbon sources. These findings imply that variations in resource availability and nutrient sources exert significant control over planktonic community composition, which is reflected by stable isotopic signatures.

Falcioni T, Manti A, Boi P, Canonico B, Balsamo M, Papa S (2006) Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry. Cytometry B Clin Cytom 70 :149-153


BACKGROUND : In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs. METHODS : Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NADS) protocol (Barbesti et al., Cytometry 2000 ;40:214-218) and on flow cytometry. RESULTS : Each method showed a good efficiency in terms of bacterial detachment ; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 x 45’’). CONCLUSIONS : The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples.

Falcioni T, Manti A, Boi P, Canonico B, Balsamo M, Papal S (2006) Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry. Cytometry Part B-Clinical Cytometry 70B :149-153

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Background : In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs.
Methods : Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NABS) protocol (Barbesti et al, Cytometry 2000 ;40:214-218) and on flow cytometry.
Results : Each method showed a good efficiency in terms of bacterial detachment ; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 x 45 ’’).
Conclusions : The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples. (c) 2006 International Society for Analytical Cytology.

Farinas B, Mary C, de OMCL, Bhaud Y, Peaucellier G, Moreau H (2006) Natural synchronisation for the study of cell division in the green unicellular alga Ostreococcus tauri. Plant Mol Biol 60 :277-292


Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.

Ferdelman TG, Thamdrup B, Canfield DE, Glud RN, Kuever J, Lillebaek R, Ramsing NB, Wawer C (2006) Biogeochemical controls on the oxygen, nitrogen and sulfur distributions in the water column of Golfo Dulce : an anoxic basin on the Pacific coast of Costa Rica revisited. Revista De Biologia Tropical 54 :171-191

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Chemical distributions, respiration rates, and bacterial distributions were measured in 1994 in the water column and sediments of a small, tropical, anoxic basin (Golfo Dulce, Pacific coast of Costa Rica) to examine the biogeochemical controls on anoxia, sulfide, dissolved inorganic nitrogen, and organic carbon consumption. As reported previously, the deepest 100 in of the water column were anoxic, and sulfide concentrations in the bottom waters were less than 7 mu M and then only transiently. Both free-swimming sulfide-oxidizing bacteria and Beggiatoa sp. (containing large vacuoles) were observed in the anoxic bottom waters or at the sediment-water interface. Aerobic respiration dominated the decomposition of organic matter in the surface waters and pycnocline, whereas sulfate reduction was principally restricted to the sediments. Bacteria were distributed in discrete zones and exhibited the highest densities where oxygen decreased below 1 mu M around 100 in depth, and near the sediment-water interface. The sub-oxic, sub-pycnocline water column was characterized by a dissolved inorganic nitrogen (DIN) deficit of 2.9 mole m(-2). With a water residence time of 35 - 57 d, estimated from a salt balance, this deficit corresponded to a DIN loss of 51 - 85 mmol m(-2) d(-1), comparable to the sub-pycnocline oxygen consumption. Sulfide in the water column was maintained at low concentrations by frequent inputs of oxygenated water from the Pacific Ocean. Sulfide production in the sediments due to bacterial sulfate reduction was scavenged by frequent deposition of iron-rich turbidites. Based on Pb-210 distributions, the most recent emplacement of a turbidite in the basin sediments was determined to have occurred between 1989 and 1992.

Ferreyra GA, Mostajir B, Schloss IR, Chatila K, Ferrario ME, Sargian P, Roy S, Prod’homme J, Demers S (2006) Ultraviolet-B radiation effects on the structure and function of lower trophic levels of the marine planktonic food web. Photochem Photobiol 82 :887-897


The impact of UV-B radiation (UVBR ; 280-320 nm) on lower levels of a natural plankton assemblage (bacteria, phytoplankton and microzooplankton) from the St. Lawrence Estuary was studied during 9 days using several immersed outdoor mesocosms. Two exposure treatments were used in triplicate mesocosms : natural UVBR (N treatment, considered as the control treatment) and lamp-enhanced UVBR (H treatment, simulating 60% depletion of the ozone layer). A phytoplankton bloom developed after day 3, but no significant differences were found between treatments during the entire experiment for phytoplankton biomass (chlorophyll a and cell carbon) nor for phytoplankton cell abundances from flow cytometry and optical microscopy of three phytoplankton size classes (picoplankton, nanoplankton and microplankton). In contrast, bacterial abundances showed significantly higher values in the H treatment, attributed to a decrease in predation pressure due to a dramatic reduction in ciliate biomass (approximately 70-80%) in the H treatment relative to the N treatment. The most abundant ciliate species were Strombidinium sp., Prorodon ovum and Tintinnopsis sp. ; all showed significantly lower abundances under the H treatment. P. ovum was the less-affected species (50% reduction in the H treatment compared with that of the N control), contrasting with approximately 90% for the other ones. Total specific phytoplanktonic and bacterial production were not affected by enhanced UVBR. However, both the ratio of primary to bacterial biomass and production decreased markedly under the H treatment. In contrast, the ratio of phytoplankton to bacterial plus ciliate carbon biomass showed an opposite trend than the previous results, with higher values in the H treatment at the end of the experiment. These results are explained by the changes in the ciliate biomass and suggest that UVBR can alter the structure of the lower levels of the planktonic community by selectively affecting key species. On the other hand, linearity between particulate organic carbon (POC) and estimated planktonic carbon was lost during the postbloom period in both treatments. On the basis of previous studies, our results can be attributed to the aggregation of carbon released by cells to the water column in the form of transparent exopolymer particles (TEPs) under nutrient limiting conditions. Unexpectedly, POC during such a period was higher in the H treatment than in controls. We hypothesize a decrease in the ingestion of TEPs by ciliates, in coincidence with increased DOC release by phytoplankton cells under enhanced UVBR. The consequences of such results for the carbon cycle in the ocean are discussed.

Fischer UR, Wieltschnig C, Kirschner AKT, Velimirov B (2006) Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms. Environmental Microbiology 8 :1394-1407

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In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h(-1) (average, 0.033 h(-1)), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF-1 h(-1)) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate(-1) h(-1)). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5-19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers.

Freeman JL, Rayburn AL (2006) Aquatic herbicides and herbicide contaminants : In vitro cytotoxicity and cell-cycle analysis. Environ Toxicol 21 :256-263


Concerns have arisen about the possible effects of herbicide contamination in aquatic ecosystems. Crop herbicides are introduced into the aquatic environment both inadvertently through runoff events and intentionally through the use of those registered for use in waterways. Acetochlor and atrazine are two agricultural crop herbicides that have often been reported to contaminate waters. Diquat and fluridone are both registered aquatic management herbicides. In this study, a mammalian in vitro cell cytotoxicity assay was used to evaluate the cytotoxicity of these four commonly used herbicides. The ranked order of the cytotoxicity was : diquat (C(1/2) = 0.036 mM +/- 0.011) > acetochlor (C(1/2) = 0.060 mM +/- 0.010) > fluridone (C(1/2) = 0.172 mM +/- 0.029) atrazine (C(1/2) = 0.581 mM +/- 0.050). In addition, flow cytometric analysis was conducted on CHO cells to investigate the potential impact of these four herbicides on the cell cycle. Acetochlor and diquat had the greatest impact on the cell cycle. Acetochor exposure resulted in a decreased number of cells in the G1 phase of the cell cycle, whereas diquat exposure resulted in a decreased number of cells in both the G1 and G2 phases. Both atrazine and fluridone resulted in a decrease in cells in the G2 phase. The agricultural crop herbicides and aquatic management herbicides gave similar results in cytotoxicity and in the cell-cycle assay at the end points tested.

Freese HM, Karsten U, Schumann R (2006) Bacterial abundance, activity, and viability in the Eutrophic River Warnow, Northeast Germany. Microbial Ecology 51 :117-127

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The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD((R)) BacLight (TM) bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4’6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker (TM) Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different states of viable and active cells in natural bacterioplankton communities. However, it remains unclear why fractions of viable and active cells were rather low in this eutrophic river in comparison to similar waters. We recommend to carefully address cells as viable in contrast to nonviable, i.e., dead. As viable cells may be active or inactive with respect to many different activities, e.g., substrate uptake, respiration, hydrolysis, and cell deviation, it is necessary to choose the method to visualize active cells according to the question to be answered.

Fuller NJ, Tarran GA, Yallop M, Orcutt KM, Scanlan DJ (2006) Molecular analysis of picocyanobacterial community structure along an Arabian Sea transect reveals distinct spatial separation of lineages. Limnology and Oceanography 51 :2515-2526

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We investigated the community structure of Synechococcus and Prochlorococcus along a transect in the Arabian Sea during September 2001. The transect spanned contrasting oceanic conditions, allowing investigation of the effects of both horizontal and vertical environmental gradients over relatively large spatial scales on picocyanobacterial population structure. We applied previously developed oligonucleotide probes specific for different Prochlorococcus ecotypes and Synechococcus clades by hybridization to ’oxygenic phototroph’ 16S ribosomal DNA polymerase chain reaction amplicons. Flow cytometry data showed that, in general, the picocyanobacterial community was dominated by Prochlorococcus in the southern oligotrophic waters and by Synechococcus in the northern mesotrophic waters. Molecular analysis of these picophytoplankton communities, however, revealed more specific spatial separation of lineages along the transect, with Prochlorococcus in southern surface waters being dominated by the high light-adapted ecotype, while low light-adapted (LL) ecotypes were confined to deeper waters below the surface-mixed layer. Interestingly, between Sta. 2 to 4, the LL genotype MIT9303 appeared to be partitioned at the very base of the euphotic zone, beneath other LL genotypes. Most of the central and northern parts of the transect were dominated by Synechococcus genotypes of the clade II lineage. A significant exception was in the mesotrophic upwelling region, where genotypes representative of clades V/VI/VII dominated. Members of the recently discovered clades IX and X were found in subsurface samples in warm, coastal waters. We propose that discrete differences, both horizontally and vertically, in a suite of environmental parameters along the transect provide optimal growth conditions for specific genotypes in a particular patch of water, giving rise to distinct, spatial compartmentalization of picocyanobacterial lineages.

Gagnaire B, Thomas-Guyon H, Burgeot T, Renault T (2006) Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes : screening of 23 molecules using flow cytometry. Cell Biol Toxicol 22 :1-14


The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks—particularly Pacific oyster, Crassostrea gigas—may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 micro mol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects.

Gerdts G, Luedke G (2006) FISH and chips : marine bacterial communities analyzed by flow cytometry based on microfluidics. J Microbiol Methods 64 :232-240


To unveil the structure of natural marine pelagic bacterial communities, PCR-based techniques as well as fluorescence in situ hybridizations (FISH) were successfully performed in the past. Using fluorescence microscopes or confocal laser scanning microscopes (CLSM) for the analysis of FISH experiments, it was possible to differentiate bacterial communities, but most attempts to combine flow cytometry and FISH for this purpose have failed till now. Here we present a successful analysis of FISH experiments of natural marine pelagic bacterial communities using a flow cytometer based on microfluidics (Agilent 2100 bioanalyzer). Marine water samples were enriched on polycarbonate filters and hybridized with Cy5 labeled gene probes of different phylogenetic depth. Bacteria were detached from the filters and subsequently analyzed in the Cell Chip of the Agilent 2100 Bioanalyzer. Samples were counter-stained using SYTOX. In all samples the EUB338 positive signals could be clearly differentiated from those of the NON probe. Furthermore a dominance of alpha-protebacteria (as indicated by the probes ALF968 and G rB) could be observed. Microfluidics based flow cytometry is a promising technique for the analysis of natural bacterial communities from the marine environment.

Gogniat G, Thyssen M, Denis M, Pulgarin C, Dukan S (2006) The bactericidal effect of TiO2 photocatalysis involves adsorption onto catalyst and the loss of membrane integrity. FEMS Microbiology Letters 258 :18-24


The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl2013KCl or sodium phosphate solutions. We found that adsorption of bacteria on the catalyst occurred immediately in NaCl2013KCl solution, whereas it was delayed in the sodium phosphate solution. We also show that the rate of adsorption of cells onto TiO2 is positively correlated with its bactericidal effect. Importantly, adsorption was consistently associated with a reduction or loss of bacterial membrane integrity, as revealed by flow cytometry. Our work suggests that adsorption of cells onto aggregated TiO2, followed by loss of membrane integrity, is key to the bactericidal effect of photocatalysis.

Hall JA (2006) Flow cytometry in aquatic ecosystem studies. Cytometry Part A 69A :403-403

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Hibi K, Abe A, Ohashi E, Mitsubayashi K, Ushio H, Hayashi T, Ren H, Endo H (2006) Combination of immunomagnetic separation with flow cytometry for detection of Listeria monocytogenes. Anal Chim Acta 573-574 :158-163


Listeria monocytogenes can grow at the low temperature commonly used in the storage and transportation of food, and the number of cases of food poisoning caused by L. monocytogenes has increased recently in the US and Europe. Several methods of detecting L. monocytogenes cells have been proposed ; however, all existing methods require approximately 48 h incubation. In this study, we attempted rapid detection of L. monocytogenes using flow cytometry (FCM). The method is based on measuring the number of L. monocytogenes cells by using a combination of FCM and immunomagnetic separation (IMS). First, polyclonal antibodies (anti-L. monocytogenes rabbit IgG-FITC) conjugated with fluorescein isothiocyanate (FITC) were reacted with L. monocytogenes cells, and then FCM was applied. The cell numbers were determined by FCM using a traditional colony-counting method in the range of 10(4)-10(8) cells ml(-1). Tetrameric antibody complexes (TAC) were used because they can recognize both magnetic and FITC molecules on the FITC-conjugated antibodies. FITC-labeled L. monocytogenes cells were reacted with a secondary antibody (TAC) bound to magnetic beads. Then, IMS was used. The method is suitable for detection in the range of 10(2)-10(8)cells ml(-1). The FCM assay enumerated the cells within 1 min and the total assay time, including sample preparation, was less than 2 h.

Hoffmann LJ, Peeken I, Lochte K, Assmy P, Veldhuis M (2006) Different reactions of Southern Ocean phytoplankton size classes to iron fertilization. Limnology and Oceanography 51 :1217-1229

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During the European Iron Fertilisation Experiment (EIFEX), performed in the Southern Ocean, we investigated the reactions of different phytoplankton size classes to iron fertilization, applying measurements of size fractionated pigments, particulate organic matter, microscopy, and flow cytometry. Chlorophyll a (Chl a) concentrations at 20-m depth increased more than fivefold following fertilization through day 26, while concentrations of particulate organic carbon (POC), nitrogen (PON), and phosphorus (POP) roughly doubled through day 29. Concentrations of Chl a and particulate organic matter decreased toward the end of the experiment, indicating the demise of the iron-induced phytoplankton bloom. Despite a decrease in total diatom biomass at the end of the experiment, biogenic particulate silicate (bPSi) concentrations increased steadily due to a relative increase of heavily silicified diatoms. Although diatoms > 20 mu m were the main beneficiaries of iron fertilization, the growth of small diatoms (2-8 mu m) was also enhanced, leading to a shift from a haptophyte- to a diatom-dominated community in this size fraction. The total biomass had lower than Redfield C : N, N : P, and C : P ratios but did not show significant trends after iron fertilization. This concealed various alterations in the elemental composition of the different size fractions. The microplankton (> 20 mu m) showed decreasing C : N and increasing N : P and C : P ratios, possibly caused by increased N uptake and the consumption of cellular P pools. The nanoplankton (2-20 mu m) showed almost constant C : N and decreasing N : P and C : P ratios. Our results suggest that the latter is caused by a shift in composition of taxonomic groups.

Hsiao A, Liu Z, Joelsson A, Zhu J (2006) Vibrio cholerae virulence regulator-coordinated evasion of host immunity. Proc Natl Acad Sci U S A 103 :14542-14547


To successfully propagate and cause disease, pathogenic bacteria must modulate their transcriptional activities in response to pressures exerted by the host immune system, including secreted immunoglobulins such as secretory IgA (S-IgA), which can bind and agglutinate bacteria. Here, we present a previously undescribed flow cytometry-based screening method to identify bacterial genes expressed in vitro and repressed during infections of Vibrio cholerae, an aquatic Gram-negative bacterium responsible for the severe diarrheal disease cholera. We identified a type IV mannose-sensitive hemagglutinin (MSHA) pilus that is repressed specifically in vivo. We showed that bacteria that failed to turn off MSHA biosynthesis were unable to colonize the intestines of infant mice in the presence of S-IgA. We also found that V. cholerae bound S-IgA in an MSHA-dependent and mannose-sensitive fashion and that binding of S-IgA prevented bacteria from penetrating mucus barriers and attaching to the surface of epithelial cells. The ability of V. cholerae to evade the non-antigen-specific binding of S-IgA by down-regulating a surface adhesin represents a previously undescribed mechanism of immune evasion in pathogenic bacteria. In addition, we found that repression of MSHA was mediated by the key virulence transcription factor ToxT, indicating that V. cholerae is able to coordinate both virulence gene activation and repression to evade host defenses and successfully colonize intestines.

Jenkins BD, Zehr JP, Gibson A, Campbell L (2006) Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments. Environmental Microbiology 8 :2083-2095

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Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment ; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai’i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.

Jiao NZ, Zhang Y, Chen Y (2006) Time series observation based InfraRed Epifluorescence Microscopic (TIREM) approach for accurate enumeration of bacteriochlorophyll-containing microbes in marine environments. Journal of Microbiological Methods 65 :442-452

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Bacteriochlorophyll a Containing Microbes (BCM) are a unique group of microorganisms in the marine environment. Accurate determination of their abundance is critical for understanding their role in energy flow and carbon cycle in the ecosystem. The InfraRed Epifluorescence Microscopy (IREM) method, using infrared fluorescence as the diagnostic signal of BCM, is the most convenient means to date for enumeration of BCM in seawater, but IREM methodology suffers from serious errors introduced by cyanobacteria, which also can emit infrared fluorescence and whose abundance is of the same order of magnitude as BCM. In the present study, an advanced "Time-series observation based cyanobacteria-calibrated InfraRed Epifluorescence Microscopy (TIREM)" approach is established for accurate enumeration of BCM in marine environments. The protocol is distinguished by its use of time series observation, auto-imaging and digital analysis. In principle, the correct count of BCM can be obtained by subtracting the cyanobacterial count from the total infrared positive count. The challenge, however, is that Prochlorococcus, the most abundant cyanobacterium in the sea, is readily visible in infrared images but not visible in the initial cyanobacterial images obtained by epifluorescence microscopy because its emission signals are masked by brighter fluorescence from larger cells like Synechococcus coexisting in seawater samples. Prochlorococcus cells become gradually visible when the fluorescence from Synechococcus cells declines after a period of exposure to excitation light. Therefore the plateau (maximum) count of the cyanobacterial cells in time series images rather than in the initial ones, as previously believed, represents the correct count for the total number of cyanobacteria (Synechococcus plus Prochlorococcus cells). Thus, the accurate estimation of BCM abundance can only be calculated from the formula : [BCM cells] = [plateau count of infrared positive cells] - [plateau count of cyanobacterial cells]. The conceptual advance of the TIREM protocol is that in classical epifluorescence microscopy or in IREM protocols, quick observation is recommended to avoid quenching the fluorescence, but in the TIREM protocol, instead, time series observation is the key for obtaining reliable data. The TIREM protocol is validated by studies using BCM and cyanobacterial pure cultures as well as by examination of samples from various marine environments. (c) 2005 Elsevier B.V. All rights reserved.

Jiao NZ, Zhao YL, Luo TW, Wang XL (2006) Natural and anthropogenic forcing on the dynamics of virloplankton in the Yangtze river estuary. Journal of the Marine Biological Association of the United Kingdom 86 :543-550

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Seasonal investigation Of virus dynamics by flow cytometry was conducted in the Yangtze river estuarine area in April, August, November 2002 and February 2003, and a supplemental Investigation in the inner estuary and downstream of the river was conducted in October 2005. The majority of the total viral abundance was bacteriophage and only 5.4% of the total was algal virus. Total viral abundance varied with season and location, ranging from 6.75x10(-5)-1.68x10(7) particles/ml, and the virus:bacterium ratio (VBR) ranged from 1.52 to 72.02 with a mean of 8.7. In the present study, viral abundance peaked in both the summer and the winter, unlike the typical seasonal pattern reported in the literature, in which viral abundance peaks in the summer when bacterial hosts are also at their most abundant. However, the driving forces for the two peaks reported here were totally different, the summer viral abundance peak coupled With the development of bacterial hosts which were controlled largely by temperature year-round and by trophic state occasionally while the winter one seemed to be multi-factor controlled. The host-phage interaction was no longer predominant in control of the winter viral abundance as bacterial abundance was lowest in this season. The winter low temperature would help maintain a high vital abundance as high temperatures might increase viral inactivation and viral decay ; the VBR peak values actually Occurred In the winter. More importantly, the high virus-containing freshwater discharge in winter due to a higher proportion Of anthropogenic sewage relative to low natural flooding in Winter run-off, turned Out to be the first Factor contributing to the high winter Viral abundance and VBR values. Ill addition, the variation of intrusion of warm and relatively oligotrophic water from oceanic Currents played a role alternating the distribution patterns of temperature, salinity and trophic conditions and consequently the distribution patterns of virus and bacteria seasonally and spatially. Dynamics of virus in the Yangtze river estuarine. rea is thus characterized by distinct seasonal and spatial variations due to natural forcing and by, and by pronounced alternation of the regular patterns due to anthropogenic Impacts.

Jorio H, Tran R, Meghrous J, Bourget L, Kamen A (2006) Analysis of baculovirus aggregates using flow cytometry. Journal of Virological Methods 134 :8-14

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Aggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures (Shen, C.F., Meghrous, J., Kamen, EA., 2002. Quantitation of baculovirus particles by flow cytometry. J. Virol. Meth. 105 (2), 321-330.). In the present study, the use of the flow cytometry method was extended to the detection of baculovirus aggregates. Flow cytometry analysis of freshly prepared baculovirus stocks, stained with SYBR Green, generally exhibited a single unimodal distribution ; while, baculovirus stocks stored at 4 degrees C for a few months exhibited a bimodal distribution of the fluorescent intensity signal. The bimodal distribution was associated with a decrease in the size of the original viral population and an emergence of a new viral population with a high fluorescence intensity. Treatment of these samples with an endonuclease (Benzonase (R)) confirmed that the new population observed in the flow cytometry analysis is not free cellular DNA. Filtration through 0.22 and 0.45 mu m membranes of the stored samples prior to flow cytometry analysis confirmed that the high fluoresecence intensity population involved particles larger than a single baculovirus. Exposing freshly amplified baculovirus stocks with a unimodal distribution to a pH of 5.3, a condition known to induce aggregation, showed the emergence of a second population with a bimodal distribution. These results suggest that flow cytometry analysis could be used to detect baculovirus aggregates. The aggregates were associated with high fluorescence intensity populations and the mean green fluorescence intensity of these populations could be used as an indicator of the mean aggregate size. Crown Copyright (c) 2005 Published by Elsevier B.V. All rights reserved.

Joux F, Agogue H, Obernosterer I, Dupuy C, Reinthaler T, Herndl GJ, Lebaron P (2006) Microbial community structure in the sea surface microlayer at two contrasting coastal sites in the northwestern Mediterranean Sea. Aquatic Microbial Ecology 42 :91-104

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In an attempt to compare the microbial community structure between the sea surface microlayer (SML) and subsurface waters (SSW), we determined the enrichment factors (EF : the ratio of abundance or activity in the SML to abundance or activity in SSW) of 13 biological parameters. Samples were taken at 2 contrasting coastal sites in the Mediterranean Sea, corresponding to a high (Barcelona, Spain) and low (Banyuls-sur-Mer, France) urbanized area. Principal component analysis showed that temporal variability was much higher at Barcelona than at Banyuls, and that the characteristics of the SML and SSW samples were more closely related at Barcelona. At both sites, the SML was weakly enriched in heterotrophic bacteria (on average 1.1-fold), Synechococcus spp. (on average 1.1-fold), and photosynthetic picoeukaryotes (on average 1.5-fold) relative to SSW. In contrast, the SML was considerably enriched in chlorophyll a (chl a) (on average 1.9-fold), phaeophytin a (on average 7.4-fold), autotrophic (on average 6.1-fold) and heterotrophic nanoflagellates (on average 5.1-fold) relative to SSW. Enrichments in bacterial production and culturable bacteria were highly variable. EF were significantly different between the 2 sites only for concentrations of chl a, b, and c and the abundance of autotrophic nanoflagellates, with higher EF at the Barcelona site. Except for autotrophic flagellates, the abundance or activity of the parameters determined in the SML was highly correlated with that determined in SSW, suggesting that enrichment of the SML results mainly from upward transport of microorganisms attached to buoyant particles or bubble scavenging. In contrast, the high contribution of autotrophic nanoflagellates to overall phytoneuston biomass (mean=26%) is likely due to their rapid colonization of the SML. The high abundances of auto- and heterotrophic nanoflagellates in the SML indicated that these organisms play a key role in the functioning of the microbial food webs at the air-sea interface.

Kalyuzhnaya MG, Zabinsky R, Bowerman S, Baker DR, Lidstrom ME, Chistoserdova L (2006) Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations. Applied and Environmental Microbiology 72 :4293-4301

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A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and Jae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISHIFC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10(4) or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

Katano T, Nakano S (2006) Growth rates of Synechococcus types with different phycoerythrin composition estimated by dual-laser flow cytometry in relationship to the light environment in the Uwa Sea. Journal of Sea Research 55 :182-190

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In the Uwa Sea, Synechococcus types with low- and high-phycourobilin (PUB) to phycoerythrobilin (PEB) ratio co-occurred throughout a year. To clarify the effects of light quality and quantity on the two types of Synechococcus, we measured the growth rates of two pigment types of Synechococcus cells with in situ incubation experiments. Incubations were conducted at 2, 10, and 20 m depth between May and October 2002. Synechococcus were divided into high- and low-PUB types using a dual-laser flow cytometer. Two indexes were used to evaluate the light environment : one was the relative light intensity (RLI) to that at the surface, and the other was the ratio of the light intensities of blue (490-500 nm) to green (540-550 nm). At 2 m depth, where the relative light intensities were above 20%, the growth rates of the low-PUB type were generally slightly higher than those of the high-PUB type. In contrast, at 10 and 20 to depth, the type that grew faster did not depend on the combination of light intensity and quality. Available light in the deeper layer of the Uwa Sea ranged from 490 to 550 nm. The range covers absorbance maxima of both PUB (ca. 495 nm) and PEB (545 nm). For this reason, light quality may not have caused one type to grow faster. These results explain the co-occurrence of two pigment types of Synechococcus in coastal waters. (c) 2005 Elsevier B.V. All rights reserved.

Kim KH, Kim YW, Kim HB, Lee BJ, Lee DS (2006) Anti-apoptotic activity of laminarin polysaccharides and their enzymatically hydrolyzed oligosaccharides from Laminaria japonica. Biotechnol Lett 28 :439-446


Laminarin polysaccharides (LP1) were prepared from Laminaria japonica, a marine brown alga with potential biological activities, by hot water extraction, ultrafiltration and gel chromatography ; the molecular weights of the LP1s were between 5 and 10 kDa. Laminarin oligosaccharides (LO) derived by hydrolyzing LP1 with an endo-beta-(1—>3)-glucanase from Bacillus circulans were mainly di- and penta-oligosaccharides. Treatment of mouse thymocytes with LO or LP1 (1-4 mg ml(-1)) suppressed apoptotic death around 3- or 2-fold and extended cell survival in culture at a rate of about 30 or 20%. A mouse cDNA microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO provided preliminary information regarding the immunomodulatory mechanism of LO. These results suggest that laminarin oligosaccharides and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines.

Kremp A, Parrow MW (2006) Evidence for asexual resting cysts in the life cycle of the marine peridinoid dinoflagellate, Scrippsiella hangoei. Journal of Phycology 42 :400-409

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Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick-walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (> 95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (< 5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.

Labreuche Y, Lambert C, Soudant P, Boulo V, Huvet A, Nicolas JL (2006) Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01/32. Microbes and Infection 8 :2715-2724

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The strategies used by bacterial pathogens to circumvent host defense mechanisms remain largely undefined in bivalve molluscs. In this study, we investigated experimentally the interactions between the Pacific oyster (Crassostrea gigas) immune system and Vibrio aestuarianus strain 01/32, a pathogenic bacterium originally isolated from moribund oysters. First, an antibiotic-resistant V. aestuarianus strain was used to demonstrate that only a limited number of bacterial cells was detected in the host circulatory system, suggesting that the bacteria may localize in some organs. Second, we examined the host defense responses to V. aestuarianus at the cellular and molecular levels, using flow-cytometry and real-time PCR techniques. We showed that hemocyte phagocytosis and adhesive capabilities were affected during the course of infection. Our results also uncovered a previously-undescribed mechanism used by a Vibrio in the initial stages of host interaction : deregulation of the hemocyte oxidative metabolism by enhancing the production of reactive oxygen species and down-regulating superoxide dismutase (Cg-EcSOD) gene expression. This deregulation may provide an opportunity to the pathogen by impairing hemocyte functions and survival. These findings provide new insights into the cellular and molecular bases of the host-pathogen interactions in C. gigas oyster. (c) 2006 Elsevier Masson SAS. All rights reserved.

Labreuche Y, Soudant P, Goncalves M, Lambert C, Nicolas JL (2006) Effects of extracellular products from the pathogenic Vibrio aestuarianus strain 01/32 on lethality and cellular immune responses of the oyster Crassostrea gigas. Developmental and Comparative Immunology 30 :367-379

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Vibrio aestuarianus strain 01/32 was previously shown to be pathogenic to Crassostrea gigas juveniles. To investigate virulence mechanisms of this pathogen, we studied the toxicity to oysters of its extracellular products (ECPs). ECPs displayed lethality to animals, with a LD50 value of 3.3 mu g/g body weight. To determine the oyster cellular immune responses induced by these ECPs, we further examined in vitro their effects on C. gigas hemocytes, using flow cytometric-based hemocyte assays. Treatment of hemolymph with ECPs caused a significant inhibition of hemocyte phagocytosis and adhesive capabilities. In contrast, the pathway of reactive oxygen species production was enhanced by higher ECP concentrations. Exposure of hemocytes to live bacteria induced no changes in hemocyte parameters. Together, these results suggest that V. aestuarianus strain 01/32 secretes one or more factors which may play an important role in the pathogenicity of this microorganism, and which display immunosuppressant activities on hemocyte functions. (c) 2005 Elsevier Ltd. All fights reserved.

Lamy D, Artigas LF, Jauzein C, Lizon F, Cornille V (2006) Coastal bacterial viability and production in the eastern English Channel : A case study during a Phaeocystis globosa bloom. Journal of Sea Research 56 :227-238

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Heterotrophic bacterial standing stocks (total and viable cells) and production were determined in the coastal surface waters of the eastern English Channel, during different stages of a phytoplankton succession. Two coastal zones of variable freshwater influence were surveyed within the ’coastal flow system’ (Wimereux and Somme Bay) where massive and recurrent Phaeocystis globosa blooms take place in spring.
The proportion of intact (MEM+) cells, assessed by the LIVE/DEAD (R) BacLight (TM) (L/D) method, varied from 15 to 94% at the two coastal stations studied (median of 46%). MEM+ and total (DAPI) cell counts were significantly correlated over the study period, whereas the higher proportion of MEM+ cells did not correspond to an elevated bacterial cell production (BP). Low levels of living (potentially active) cells were nevertheless responsible for the high productivity levels within the bacterial community when the P. globosa bloom declined. Our study revealed that the bacterial carbon production/primary production ratios (BCP/PP) showed broad variations (7 to 111%) within each site, going from low values (7-16%) when the bloom was the most productive, to higher values (61-111%) at the end of the bloom. This suggested (i) a temporal uncoupling between bacteria and phytoplankton throughout the bloom duration and (ii) a drastic change of the amount of PP potentially processed by the bacterial community among high and low productive periods. The BCP increase after the decline of the P globosa bloom implies that, at this time, a large part of the phytoplankton-derived organic matter (OM) was remineralised via the bacterial heterotrophic production. With respect to the L/D results, this bacterial remineralisation was due to a small yet productive total cell fraction. (c) 2006 Elsevier B.V All rights reserved.

Lankoff A, Bialczyk J, Dziga D, Carmichael WW, Gradzka I, Lisowska H, Kuszewski T, Gozdz S, Piorun I, Wojcik A (2006) The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor. Mutagenesis 21 :83-90


The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.

Liu LP, Wu CG, Cben TY, Zhang XJ, Li FH, Luo W, Xiang JH (2006) Effects of infection of EGH-expressing Escherichia coli on haemocytes in Ciona intestinalis. Journal of Experimental Marine Biology and Ecology 332 :121-134

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The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for Immoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage I h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians. (c) 2005 Elsevier B.V. All rights reserved.

Liu R, Zhu W, Zhang Y, Zhu T, Liu H, Fang Y, Gu Q (2006) A new diphenyl ether from marine-derived fungus Aspergillus sp. B-F-2. J Antibiot (Tokyo) 59 :362-365


A new diphenyl ether dimethyl 2,3’-dimethylosoate (1) together with three known compounds monomethylsulochrin (2), emodin (3), and questin (4) were isolated through bioassay-guided fractionations from the culture of a marine-derived fungus Aspergillus sp. B-F-2. The structures of these compounds were determined by spectroscopic methods. Cytotoxicities of compounds 1 and 2 against K562 cell line were preliminarily evaluated by the MTT method and flow cytometry.

Liu R, Zhu WM, Zhang YP, Zhu TJ, Liu HB, Fang YC, Gu QQ (2006) A new diphenyl ether from marine-derived fungus Aspergillus sp B-F-2. Journal of Antibiotics 59 :362-365

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A new diphenyl ether dimethyl 2,3 ’ dimethylosoate (1) together with three known compounds monomethylsulochrin (2), emodin (3), and questin (4) were isolated through bioassay-guided fractionations from the culture of a marine-derived fungus Aspergillus sp. B-F-2. The structures of these compounds were determined by spectroscopic methods. Cytotoxicities of compounds 1 and 2 against K562 cell line were preliminarily evaluated by the MTT method and flow cytometry.

Liu YQ, Yao TD, Kang SC, Jiao NZ, Zeng YH, Shi Y, Luo TW, Jing ZF, Huang SJ (2006) Seasonal variation of snow microbial community structure in the East Rongbuk glacier, Mt. Everest. Chinese Science Bulletin 51 :1476-1486

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The bacterial diversity and abundance in the snow of East Rongbuk glacier, Mt. Everest were examined through 16S rRNA gene clone library and flow cytometry approaches. In total, 35 16S rRNA gene sequences were obtained, which belong to alpha, beta, gamma-Proteobacteria, Actinobacteria, Firmicutes, CFB, Cyanobacteria, Eukaiyotic chloroplast, and TM7 candidate phylum respectively. gamma-Proteobacteria was the dominant bacterial group in this region, while the genera Acinetobacter and Leclercia were dominant on the genus level. The community structure varied seasonally. The bacterial abundance in summer snow was higher than that in winter. Moreover, the snow bacterial community structures in both seasons were diverse, with not only common species but season-specific species. The common species most likely originated from the Tibet Plateau. Bacteria in summer snow are affiliated with marine environment, whereas bacteria in winter snow are closely related to more diverse environments and show the feature of resistance to cold. Seasonal variations of abundance and bacterial diversity were most probably due to the seasonal characteristics of climate and atmospheric circulation in Mt. Everest.

Longnecker K, Homen DS, Sherr EB, Sherr BF (2006) Similar community structure of biosynthetically active prokaryotes across a range of ecosystem trophic states. Aquatic Microbial Ecology 42 :265-276

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Variability in both the abundance and phylogenetic diversity of biosynthetically active prokaryotes has implications for global carbon cycling. In the present study, our primary goal was to determine the extent of variability in phylogenetic diversity of biosynthetically active prokaryotes from 3 regions in the California Current System off the Oregon coast, ranging from eutrophic shelf to oligotrophic basin. Assimilation of H-3-leucine, as determined by microautoradiography, was combined with fluorescence in situ hybridization (MICROFISH) to identify biosynthetically active prokaryotes. Oligonucleotide probes targeted 2 domains (Bacteria and Archaea), and 4 groups within the Bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-like cells). We found that the Alphaproteobacteria and Cytophaga-like cells comprised the largest proportion of bacterial cells assimilating leucine. Alphaproteobacteria was the only group in which the abundance of active cells was significantly correlated to in situ phytoplankton stocks. Archaea were present in low numbers in most samples. However, in deep (> 250 m) samples from the oligotrophic basin station, 43% of cells identified as Archaea were biosynthetically active. In general, we observed a similar change in the proportional abundance of cells assimilating leucine for all identified phylogenetic groups. Thus, at this phylogenetic level, our data set is evidence for tandem increase or decrease in biosynthetic activity by the whole prokaryotic community, rather than for shifts in activity by specific phylogenetic groups.

Longnecker K, Sherr BF, Sherr EB (2006) Variation in cell-specific rates of leucine and thymidine incorporation by marine bacteria with high and with low nucleic acid content off the Oregon coast. Aquatic Microbial Ecology 43 :113-125

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The balance between rates of protein synthesis (measured by incorporation of radioactively labeled leucine) and rates of DNA synthesis (measured by incorporation of radioactively labeled thymidine) has been used to evaluate growth state in marine bacterioplankton. Our objective was to determine if variability in leucine and thymidine incorporation could further elucidate ecological differences in the role of high nucleic acid (HNA) and low nucleic acid (LNA) cells in marine ecosystems. We report here the first data set in which cell-specific rates of leucine and thymidine incorporation have been compared for HNA and LNA bacterial cells in the open ocean. In general, HNA cells had higher cell-specific incorporation rates of both leucine and thymidine, and had higher leucine:thymidine (Leu:TdR) incorporation ratios than LNA cells. The higher Leu:TdR ratios for HNA cells suggest that increases in the ratio may be associated with increased metabolic activity by marine bacterioplankton. Leu:TdR incorporation ratios were significantly positively correlated to temperature, but not to chlorophyll a concentrations or phytoplankton biomass calculated from specific carbon:chlorophyll a ratios, indicating that in this region temperature had a greater effect on bacterial growth state than did substrate supply. The proportion of total heterotrophic bacterial activity attributable to LNA cells was significantly greater at slope and basin stations compared to the mesotrophic shelf station. Finally, the difference in incorporation rates between HNA and LNA cells was greater for protein synthesis than for DNA synthesis.

Lopez-Flores R, Boix D, Badosa A, Brucet S, Quintana XD (2006) Pigment composition and size distribution of phytoplankton in a confined Mediterranean salt marsh ecosystem. Marine Biology 149 :1313-1324

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Pigment composition and size distribution of phytoplankton were analysed in a group of Mediterranean salt marshes, where hydrology is dominated by sudden inputs during sea storms, followed by long periods of confinement. These marshes are characterized by a low inorganic-organic nutrient ratio, and inorganic nitrogen is especially scarce due to denitrification. Nutrients were the main factor affecting phytoplankton biomass, while zooplankton grazing did not control either phytoplankton community composition, or their size distribution. The relative abundance of the different phytoplankton groups was analysed by correspondence analysis using the pigment composition measured by high-performance liquid chromatography (HPLC) and analysed with the CHEMTAX programme. In this analysis, phytoplankton pigment composition was correlated with two nutrient gradients. The first gradient was the ratio of nitrate-total nitrogen (TN), since the different phytoplankton groups were distributed according to their eco-physiological differences in nitrogen uptake. The second gradient was correlated with total nutrient loading. Biomass size distributions frequently showed a lack of intermediate sized nanophytoplankton (2.5-4 mu m in diameter), and the importance of this lack of intermediate sizes correlated with dinoflagellate biomass. These results suggested that in confined environments, where nutrients are mainly in an organic form, dinoflagellates take advantage of their mixotrophy, by competing and grazing on smaller phytoplankters simultaneously.

Lurling M, Roessink I (2006) On the way to cyanobacterial blooms : Impact of the herbicide metribuzin on the competition between a green alga (Scenedesmus) and a cyanobacterium (Microcystis). Chemosphere 65 :618-626

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The hypothesis that exposure to a common and widely applied photosynthesis-inhibiting herbicide, metribuzin, would alter the outcome of the competitive battle between susceptible green algae (Scenedesmus obliquus) and tolerant cyanobacteria (Microcystis aeruginosa) was tested. In a long-term (17 d) experiment, Scenedesmus and Microcystis populations as well as mixtures that started with different inoculum composition (i.e. 3:1, 1:1 and 1:3 Scenedesmus:Microcystis) were grown in the absence or presence of metribuzin (100 mu g l(-1)). In the absence of metribuzin, Scenedesmus was competitively superior and out-competed Microcystis regardless the initial composition of the mixed communities. However, this competitive outcome was reversed completely in the presence of metribuzin, where despite growth inhibition Microcystis became dominant. Hence, photosynthesis-inhibiting herbicides may not only affect algal community structure, but also provide cyanobacteria founder populations a window for dominance and thus play an important role in promoting cyanobacteria blooms. (c) 2006 Elsevier Ltd. All rights reserved.

Mai-Prochnow A, Webb JS, Ferrari BC, Kjelleberg S (2006) Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms. Appl Environ Microbiol 72 :5414-5420


In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a DeltaAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC4(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from DeltaAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the DeltaAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.

Manini E, Danovaro R (2006) Synoptic determination of living/dead and active/dormant bacterial fractions in marine sediments. Fems Microbiology Ecology 55 :416-423

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The most widely used methods for the estimation of the living/dead fractions of bacterial cells involve specific stains that are able to reveal membrane integrity. Here, we have compared two different probes (propidium iodide and ethidium homodimer-2) that have different molecular weights and steric hindrance effects. We have also combined this method with the staining/destaining procedure that is currently used in the identification of potentially active cells. The procedure for marine sediments described here allows the synoptic (i.e. from the same filter) identification of : (i) the number of living bacteria ; (ii) the number of active vs. dormant cells within this living fraction ; (iii) the bacterial fraction with an intact nucleoid region without membrane integrity ; and (iv) dead cells (devoid of the nucleoid region and without membrane integrity). Our results demonstrate that the concentration of propidium is crucial for the correct estimation of the dead bacterial fraction, ethidium homodimer-2 allows efficient and accurate estimates that are independent of the concentrations used and the sample storage. The active bacterial fraction represented c. 40% of the total bacterial abundance, the inactive/dormant fraction c. 30%, and the dead fraction was, on average, c. 30%. This method allows the processing of a large number of samples with high precision and at relatively low cost, and thus it provides additional synoptic insights into the metabolic state of bacteria in marine sediments.

Marie D, Zhu F, Balague V, Ras J, Vaulot D (2006) Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR). FEMS Microbiol Ecol 55 :403-415


The composition and abundance of eukaryotic picoplankton (defined here as cells smaller than 3 mum) was investigated in the Morocco upwelling and throughout the Mediterranean Sea in late summer using flow cytometry and molecular methods (gradient gel electrophoresis and quantitative PCR). The picoplankton displayed characteristics typical of oligotrophic oceanic areas with concentrations down to 1000 cells mL(-1) in the Eastern Basin. The most abundant eukaryotic sequences recovered by gradient gel electrophoresis were related to uncultivated marine groups : alveolates I (16%) and II (26%) and a newly discovered group (env Nansha, 17%) for which sequences have been recently obtained from the South China Sea and that could be related to Acantharians. Prasinophyceae (photosynthetic green algae) accounted for 10% of the sequences, whereas Cercozoa, Stramenopiles, Polycystinea, dinoflagellates and ciliates provided minor contributions. The use of quantitative PCR coupled with taxon-specific primers allowed us to estimate the relative abundance of several taxa belonging to the Prasinophyceae. Of the three genera assessed, Bathycoccus appeared as the most abundant, forming localized maxima at depth.

Martens-Habbena W, Sass H (2006) Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension. Applied and Environmental Microbiology 72 :87-95

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The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, How cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DA-PI [4’,6’-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and 11) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 X 10(8) Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.

Mary I, Cummings DG, Biegala IC, Burkill PH, Archer SD, Zubkov MV (2006) Seasonal dynamics of bacterioplankton community structure at a coastal station in the western English Channel. Aquatic Microbial Ecology 42 :119-126

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An annual study of the bacterioplankton community structure was carried out at Stn L4 (50 degrees 15’N, 04 degrees 13’W) in the western English Channel between August 2003 and July 2004. Bacterioplankton abundance and community structure were assessed using flow cytometry and fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, respectively. The Eubacteria domain dominated over the Archaea domain (< 15 %) at the highest phylogenetic level. The Sphingo-bacteria-Flavobacteria group of the Bacteroidetes phylum (SFB) numerically dominated in spring and early summer. The alpha-Proteobacteria dominated from late summer to winter. The SAR11 clade represented similar to 13% of the microbial community throughout the year and accounted for up to 69% of a-Proteobacteria in late spring. Annually, gamma-Proteobacteria were 2 or 3 times less abundant than the other groups and showed no obvious seasonal trend. The SAR86 cluster accounted for up to half of gamma-Proteobacteria when it peaked in summer. Consequently, we found that community structure at higher taxonomic level did not change dramatically with season but lower level phylogenetic groups showed pronounced seasonal peaks.

McBeath AJ, Collet B, Paley R, Duraffour S, Aspehaug V, Biering E, Secombes CJ, Snow M (2006) Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus. Virus Res 115 :176-184


Infectious salmon anaemia virus (ISAV) is an orthomyxovirus and member of the genus Isavirus, which contains eight genomic segments coding for ten viral proteins. This study focussed on identifying the function of the largest protein encoded by ISAV genomic segment 7 (7i), which like influenza A segment 7 encodes two proteins, one of which is based on removal of an intron from the primary transcript. Using two independent methods, an Mx1 promoter-driven reporter system and real-time PCR of FACS-sorted transfected cells, we demonstrate that the non-structural ISAV 7i protein is an interferon-signalling antagonist. Other transfection studies indicated a predominantly cytoplasmic localisation of the expressed protein, which is consistent with this role. The demonstration that ISAV segment 7 encodes a putative non-structural IFN system antagonist reveals a difference with influenza A virus, where segment 7, which shares a similar coding strategy, encodes the structural matrix proteins.

Mowlem M, Chavagnac V, Statham P, Burkill P, Benazzi G, Holmes D, Morgan H, Haas C, Kraft M, Taberham A (2006) Micro system technology for marine measurement. Oceans 2006, Vols 1-4 :845-850

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To date the development of in situ chemical and biological sensors has focused on the production of macro instruments for single point deployment. With the exception of oxygen sensors[1], [2] and biological sensors based on fluorometry [1] (that are now commercially available) chemical and biological sensors are not able to make repeated synoptic measurements at finer temporal and spatial. scales. This is at odds with the patchiness and temporal variability observed in biogeochemical processes [4]. This paper describes the early stages of development of miniature and mass producible chemical and biological sensors using Micro System Technology. It is hoped that these devices will he suitable for mass deployment and will deliver repeated synoptic data that will allow greater understanding and improved modelling of biogeochemical processes. Initially the production of two devices has been targeted : 1) A miniature cytometer to count and speciate phytoplankton ; and 2) A lab-on-a-chip analyser using wet chemistry and optical detection. The production of the analysis chip for the cytometer and a micropump suitable for the lab-on-a-chip analyser are presented here. Preliminary investigations of biofouling are also discussed.

Mukhanov VS, Naidanova OG, Lopukhina OA, Kemp RB (2006) Cell-, biovolume- and biosurface-specific energy fluxes through marine picoplankton as a function of the assemblage size structure. Thermochimica Acta 458 :23-33

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The heat production of natural heterotrophic picoplankton collected in Sevastopol Bay (the Black Sea) and its long-term (from I to 105 days) enrichment batch-culture isolated from the same site was measured by isothermal microcalorimetry. Over the period of senescence of the culture, cell miniaturisation took place, with the average cell volume decreasing from 1.09 +/- 0.15 (95% CI) to 0.18 +/- 0.02 mu m(3). For the same time period, the heat fluxes decreased from 45 +/- 3 fW per cell, 56 +/- 13 fW mu m(-3) and 10 +/- 3 fW mu m(-2) to 0.5 +/- 0.2 fW per cell, 2.1 +/- 1.1 fW mu m(-3) and 0.2 +/- 0.1 fW mu m(-2), thus providing evidence of the positive dependence of the fluxes on the cell size (r(2) = 0.45, n = 68). In the natural assemblage, biovolume- and biosurface-specific heat fluxes insignificantly (r(2) = 0.19 and 0.12, respectively ; n = 25) increased with decreasing average cell size from 0.75 +/- 0.12 to 0.13 +/- 0.04 mu m(3), to give indirect evidence that at least a part of the ultramicrobacterial pool are cells with high volume-specific metabolic rate. The maximum biosurface-specific metabolic rate measured for the natural bacteria proved to be close to those averaged for actively growing aquatic protozoans at 1.3 x 10(-15) mol O-2 mu m(-2) h(-1) (equivalent to 2 x 10(-13) W mu m(-2) for purely aerobic metabolism), as calculated from published data. The latter does not depend on the cell volume (r(2) < 0.001, n = 58) over the size range from 10(2) (smallest flagellates) to 10(8) mu m(3) (largest sarcodines), supplying illustrative evidence for Rubner’s law. Marine bacteria (10(-1) mu m(3)) appear to fit this law and extend its scale by 2 orders of magnitude. (C) 2007 Elsevier B.V. All rights reserved.

Nishimura M, Shimakita T, Kamiya E, Tashiro Y, Kogure K (2006) Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria. Fisheries Science 72 :723-727

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The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.

Ono T, Park KS, Ueta M, Iida T, Honda T (2006) Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1. Infect Immun 74 :1032-1042


Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

Paniagua-Chavez CG, Jenkins J, Segovia M, Tiersch TR (2006) Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes. Cryobiology 53 :128-138


Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P=0.004) and thawing treatments (P=0.04), and mitochondrial membrane potential (P=0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P=0.0258) and with mitochondrial membrane potential (P=0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P=0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.

Sakr S, Jeanjean R, Zhang C-C, Arcondeguy T (2006) Inhibition of Cell Division Suppresses Heterocyst Development in Anabaena sp. Strain PCC 7120. J Bacteriol 188 :1396-1404


When the filamentous cyanobacterium Anabaena PCC 7120 is exposed to combined nitrogen starvation, 5 to 10% of the cells along each filament at semiregular intervals differentiate into heterocysts specialized in nitrogen fixation. Heterocysts are terminally differentiated cells in which the major cell division protein FtsZ is undetectable. In this report, we provide molecular evidence indicating that cell division is necessary for heterocyst development. FtsZ, which is translationally fused to the green fluorescent protein (GFP) as a reporter, is found to form a ring structure at the mid-cell position. SulA from Escherichia coli inhibits the GTPase activity of FtsZ in vitro and prevents the formation of FtsZ rings when expressed in Anabaena PCC 7120. The expression of sulA arrests cell division and suppresses heterocyst differentiation completely. The antibiotic aztreonam, which is targeted to the FtsI protein necessary for septum formation, has similar effects on both cell division and heterocyst differentiation, although in this case, the FtsZ ring is still formed. Therefore, heterocyst differentiation is coupled to cell division but independent of the formation of the FtsZ ring. Consistently, once the inhibitory pressure of cell division is removed, cell division should take place first before heterocyst differentiation resumes at a normal frequency. The arrest of cell division does not affect the accumulation of 2-oxoglutarate, which triggers heterocyst differentiation. Consistently, a nonmetabolizable analogue of 2-oxoglutarate does not rescue the failure of heterocyst differentiation when cell division is blocked. These results suggest that the control of heterocyst differentiation by cell division is independent of the 2-oxoglutarate signal.

Sandaa RA, Larsen A (2006) Seasonal variations in virus-host populations in Norwegian coastal waters : focusing on the cyanophage community infecting marine Synechococcus spp. Appl Environ Microbiol 72 :4610-4618


Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system : Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 x 10(4) Synechococcus cells ml(-1) and 7.2 x 10(3) cyanophage ml(-1). By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.

Seymour JR, Seuront L, Doubell M, Waters RL, Mitchell JG (2006) Microscale patchiness of virioplankton. Journal of the Marine Biological Association of the United Kingdom 86 :551-561

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The microscale spatial distributions of viruses were investigated in three contrasting environments including oligotrophic open ocean, eutrophic coastal and estuarine habitats. The abundances or two discrete populations of both viruses and heterotrophic bacteria were measured at spatial resoulutions Of between 1 and 5 cm using purpose-designed microscale sampling equipment and flow cytometric Sample analysis. Within open water samples, virus distributions were characterized by non-normal distributions and by ’hotspots’ in abundance where concentrations varied by up to 17-fold. In contrast to patterns generally observed at larger spatiotemporal scales, there was no correlation between bacterial and viral abundance or correspondence between bacteria and virus hotspots within these samples. Consequently, strong hotspots and gradients in the virus:bacteria ratio (VBR) were also apparent within samples. Within vertical profiles taken from above the sediment-water interface within a temperate mangrove estuary, distributions of planktonic viruses were characterized by gradients in abundance, with highest concentrations observed within the 1-2 cm immediately above the sediment surface, and virus distributions were correlated to bacterial abundance (P < 0.01). The patterns observed in these contrasting habitats indicate that microscale patchiness of virus abundance may be a common feature of the marine environment. This form of heterogeneity may have important implications for virus-host dynamics and subsequently influence microbial trophodynamics and nutrient cycling in the ocean.

Shao B, Jaffe JS, Chachisvilis M, Esener SC (2006) Angular resolved light scattering for discriminating among marine picoplankton : modeling and experimental measurements. Optics Express 14 :12473-12484

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In order to assess the capability to optically identify small marine microbes, both simulations and experiments of angular resolved light scattering (ARLS) were performed. After calibration with 30-nm vesicles characterized by a nearly constant scattering distribution for vertically polarized light (azimuthal angle= 90 degrees), ARLS from suspensions of three types of marine picoplankton (two prokaryotes and one eukaryote) in seawater was measured with a scattering device that consisted of an elliptical mirror, a rotating aperture, and a PMT. Scattered light was recorded with adequate signal-to-noise in the 40-140 degrees. Simulations modeled the cells as prolate spheroids with independently measured dimensions. For the prokaryotes, approximated as homogeneous spheroids, simulations were performed using the RM (Rayleigh-Mie) - I method, a hybrid of the Rayleigh-Debye approximation and the generalized Lorentz-Mie theory. For the picoeukaryote, an extended RM - I method was developed for a coated spheroid with different shell thickness distributions. The picoeukaryote was then modeled as a coated sphere with a spherical core. Good overall agreements were obtained between simulations and experiments. The distinctive scattering patterns of the different species hold promise for an identification system based on ARLS.

Sherr EB, Sherr BF, Longnecker K (2006) Distribution of bacterial abundance and cell-specific nucleic acid content in the Northeast Pacific Ocean. Deep-Sea Research Part I-Oceanographic Research Papers 53 :713-725

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We tested the idea that bacterial cells with high nucleic acid content (HNA cells) are the active component of marine bacterioplankton assemblages, while bacteria with low nucleic acid content (LNA cells) are inactive, with a large data set (> 1700 discrete samples) based on flow cytometric analysis of bacterioplankton in the Northeast Pacific Ocean off the coast of Oregon and northern California, USA. Samples were collected in the upper 150 m of the water column from the coast to 250 km offshore during 14 cruises from March 2001 to September 2003. During this period, a wide range of trophic states was encountered, from dense diatom blooms (chlorophyll-a concentrations up to 43 mu g l(-1)) at shelf stations during upwelling season (March-September) to lower chlorophyll-a concentrations (0.1-5 mu g l(-1)) during winter (November-February) and at basin stations (> 1700 m depth). We found only weakly positive relations of log total bacterial abundance to log chlorophyll-a concentration (as a proxy for availability of organic substrate), and of HNA bacteria as a fraction of total bacteria to log chlorophyll-a. Abundance of HNA and LNA bacteria co-varied positively in all regions, although HNA bacteria were more responsive to high phytoplankton biomass in shelf waters than in slope and basin waters. Since LNA cell abundance in general showed responses similar to those of HNA cell abundance to changes in phytoplankton biomass, our data do not support the hypothesis that HNA cells are the sole active component of marine bacterioplankton. (c) 2006 Elsevier Ltd. All rights reserved.

Shibata A, Goto Y, Saito H, Kikuchi T, Toda T, Taguchi S (2006) Comparison of SYBR Green I and SYBR Gold stains for enumerating bacteria and viruses by epifluorescence microscopy. Aquatic Microbial Ecology 43 :223-231

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SYBR Gold staining is used for enumerating bacteria and viruses in aquatic samples. However, its suitability for epifluorescence microscopy has not been sufficiently investigated. Thus we compared bacterial and viral counts using SYBR Gold and SYBR Green I stains. Variables for both bacterial and viral counts included season and ocean depths of sample collection and the period of sustained excitation under epifluorescence microscopy. We also examined the storage period and procedures for preservation of samples with formaldehyde for bacterial counts. Natural seawater samples were used for all experiments. Ratios of counts obtained with SYBR Gold to those with SYBR Green I staining were 0.99 +/- 0.09 (mean +/- SD, n = 58) for bacteria and 1.0 +/- 0.1 (n = 38) for viruses, which indicated no significant differences between stains. In samples fixed with 0.74 % formaldehyde that were stored at 4 degrees C, bacterial counts obtained with SYBR Gold staining decreased over time in parallel with those obtained with SYBR Green I staining. However, counts from fixed samples with both SYBR stains did not decrease significantly after 30 d when glass slides were prepared immediately and stored at -20 degrees C, or when samples were flash-frozen in liquid nitrogen and stored at -80 degrees C. Under sustained excitation, counts of bacteria and viruses stained with SYBR Gold decreased less than with SYBR Green 1, suggesting greater persistence of the fluorescence signal with SYBR Gold. These results indicate the suitability of SYBR Gold staining for use in the determination of bacterial and viral abundance in natural seawater.

Sintes E, Herndl GJ (2006) Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with micro autoradiography. Applied and Environmental Microbiology 72 :7022-7028

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Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking Up H-3-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.

Stabili L, Licciano M, Giangrande A, Longo C, Mercurio M, Marzano CN, Corriero G (2006) Filtering activity of Spongia officinalis var. adriatica (Schmidt) (Porifera, Demospongiae) on bacterioplankton : Implications for bioremediation of polluted seawater. Water Research 40 :3083-3090

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A study on the filtering activity has been carried out on reared specimens of the demosponge Spongia officinalis var. adriatica coming from an off-shore farm displaced off the Apulian coast (Ionian Sea). The experience was carried out under laboratory conditions, by using natural seawater collected from the sponge environment. The study demonstrates a high efficiency of the sponge in removing bacteria. Bacterial concentration significantly decreases in presence of the sponge, with a marked drop after 2h from the start of the experience. The maximum clearance rate was 210 ml h(-1) g(-1) DW at 60 min. Retention efficiency reached the highest value of 61% at 120 min. The bacterial density removed by the S. officinalis filtering activity was 12.3 +/- 1.8 x 10(4) cells ml(-1) corresponding to a biomass of about 11.7 +/- 1.4 mu g Cl-1. The sponge fed preferentially large- and medium-size bacteria, whereas the small ones are fed after the removal of the largest size categories. The results obtained suggest that S. offcinalis is a suitable species for marine environmental bioremediation. (c) 2006 Elsevier Ltd. All rights reserved.

Staropoli A, Baudouin C, Dutot M, Sultan G, Warnet JM, Rat P (2006) Role of conditioning medium cytotoxicity. An example with an intraocular lens preloaded in injector. Journal Francais D Ophtalmologie 29 :773-780

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Introduction : Following material vigilance cases encountered with the hydrophilic acrylic intraocular lens, ACR6D SE preloaded in the Premier(R) shooter, we studied the cytotoxicity of the intraocular lens and its conditioning to identify the cytotoxic element. We proposed medical device modification to improve its biocompatibility.
Materials and Methods : Biocompatibility-cytotoxicity assays were carried out according to ISO 10993-5 recommendations. Tests were performed on the SRA 01/04 human lens epithelial cell line. Neutral red, Hoechst 33342, and YO-PRO-1 fluorescent probes were used to assess membrane integrity, total DNA, and membrane fluidity, respectively. Materials samples were prepared in culture medium according to the ISO 10993-5 elution procedure. Pure saline solutions and conditioning liquids were tested directly on cells.
Results : The intraocular lens and injector were not cytotoxic. Conditioning liquids induced membrane fluidity perturbation characteristic of apoptosis. Tests performed on new versions of the medical device identified a better adapted conditioning liquid.
Conclusion : The results suggest that the cytotoxicity of the conditioning liquid could explain the postoperative complication rate. When we changed the conditioning liquid with sterile irrigating solution (i.e., rich divalent cation marine solution), we eliminated cellular stress. Fluorescent probes are well adapted to assess medical device biocompatibility-cytotoxicity.

Topping JN, Heywood JL, Ward P, Zubkov MV (2006) Bacterioplankton composition in the Scotia Sea, Antarctica, during the austral summer of 2003. Aquatic Microbial Ecology 45 :229-235

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Physical ocean processes (ice-melt, island run-off and upwelling of nutrients) were hypothesised to affect the bacterioplankton composition in the surface mixed layer of the Scotia Sea during the austral summer of 2003, and this was investigated using flow cytometry and catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH) techniques. The bacterioplankton was composed predominantly of Alphaproteobacteria (PB), comprising SAR11, Roseobacter spp. and SAR116 groups, followed by Sphingobacteria/Flavobacteria and Gammaproteobacteria, including SAR86. Two distinct bacterioplankton communities were identified, largely based on bacterioplankton abundance, which varied from 0.3 +/- 0.06 x 10(6) cells ml(-1) in the west to 0.8 +/- 0.3 x 10(6) cells ml(-1) in the east, and a corresponding difference in SAR11 percentages of 30 +/- 15% in the west compared to 5 +/- 5% in the east. The western community was present in waters that were largely in an over-wintered, pre-bloom condition. The eastern bacterioplankton community was associated with phytoplankton blooms developed within the eastern Scotia Sea nutrient upwelling zone, where the Antarctic Circumpolar Current (ACC) encounters the shallow bathymetry associated with the Scotia Arc, in combination with seasonal ice-melt and island effects that enabled surface water stratification.

Uysal Z (2006) Vertical distribution of marine cyanobacteria Synechococcus spp. in the Black, Marmara, Aegean, and eastern Mediterranean seas. Deep-Sea Research Part Ii-Topical Studies in Oceanography 53 :1976-1987

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The vertical distributions of the unicellular cyanobacteria Synechococcus were studied in several highly contrasting seas : the Black Sea, Sea of Marmara, Aegean Sea, and Mediterranean Sea. Cell abundances varied significantly on both vertical and horizontal scales in all physically and spatially discrete water masses. Epifluorescence microscope cell counts from all seas clearly showed that majority of the population remains suspended in the surface-mixed layer and decreases gradually towards the base of the euphotic zone. Surface spatial distributions in the Black Sea were heterogeneous. Salinity, rather than temperature, seemed to have the greatest impact on the surface distribution of cells in this highly eutrophic sea. Changes in abundance in the mixed layer were small compared to the abrupt changes below the halocline, especially in the Black Sea and the Sea of Marmara. In contrast to the Black Sea, the major population remains suspended above the depth of fluorescence maximum in the Aegean and eastern Mediterranean seas. Significant correlations (r > P-0.01) were observed between cell counts and physical and chemical parameters with depth in the Black Sea. In all seas, cells at subsurface chlorophyll-a maximum layer (SCML) reflected brighter and longer fluorescence than those present at the surface and below. Cell size derived from flow cytometry indicated the presence of larger cells at the surface mixed layer compared to those at depth. (c) 2006 Elsevier Ltd. All rights reserved.

Verspagen JMH, Passarge J, Johnk KD, Visser PM, Peperzak L, Boers P, Laanbroek HJ, Huisman J (2006) Water management strategies against toxic Microcystis blooms in the Dutch delta. Ecological Applications 16 :313-327

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To prevent flooding of the Dutch delta, former estuaries have been impounded by the building of dams and sluices. Some of these water bodies, however, experience major ecological problems. One of the problem areas is the former Volkerak estuary that was turned into,a freshwater lake in 1987. From the early 1990s onward, toxic Microcystis blooms dominate the phytoplankton of the lake every summer. Two management strategies have been suggested to suppress these harmful algal blooms : flushing the lake with fresh water or reintroducing saline water into the lake. This study aims at an advance assessment of these strategies through the development of a mechanistic model of the population dynamics of Microcystis. To calibrate the model, we monitored the benthic and pelagic Microcystis populations in the lake during two years. Field samples of Microcystis were incubated in the laboratory to estimate growth and mortality rates as functions of light, temperature, and salinity. Recruitment and sedimentation rates were measured in the lake, using traps, to quantify benthic-pelagic coupling, of the Microcystis populations. The model predicts that flushing with fresh water will suppress Microcystis blooms when the current flushing rate is sufficiently increased. Furthermore, the inlet of saline water will suppress Microcystis blooms for salinities exceeding 14 g/L. Both management options are technically feasible. Our study illustrates that quantitative ecological knowledge can be a helpful tool guiding large-scale water management.

Wada M, Kagami Y, Ogata N, Horinouchi S (2006) Preparation of DNA-polymer microsphere and its optical property - art. no. 60370H. Device and Process Technologies for Microelectronics, MEMS, and Photonics IV 6037 :H370-H370

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We succeeded to prepare the microsphere of polymer to which the marine DNA was doped. Moreover, ASE (Amplified Spontaneous Emission) was observed since the optical property of the microsphere to which the DNA was doped was discussed. In addition, an excellent optical property was found being shown in the comparison with DNA doped polymer thin film. The DNA shows a very interesting optical property like the improvement of the fluorescent intensity and the control of the concentration quenching of the dye by inserting the functional dye between the base pairs. On the other hand, the research using the surface is advanced to the microsphere for the reasons why the surface area is large. Light can be confined highly effective in the miniature space, and the microsphere of polymer is a material that is promising as optical micro cavity.

Wu C, Yang F (2006) Localization studies of two white spot syndrome virus structural proteins VP51 and VP76. Virol J 3 :76


VP51 and VP76 are two structural proteins of white spot syndrome virus (WSSV). However, there is some controversy about their localization in the virion at present. In this study, we employ multiple approaches to reevaluate the location of VP51 and VP76. Firstly, we found VP51 and VP76 presence in viral nucleocapsids fraction by Western blotting. Secondly, after the high-salt treatment of nucleocapsids, VP51 and VP76 were still exclusively present in viral capsids by Western blotting and immunoelectron microscopy, suggesting two proteins are structural components of the viral capsid. To gather more evidence, we developed a method based on immunofluorescence flow cytometry. The results revealed that the mean fluorescence intensity of the viral capsids group was significantly higher than that of intact virions group after incubation with anti-VP51 or anti-VP76 serum and fluorescein isothiocyanate conjugated secondary antibody. All these results indicate that VP51 and VP76 are both capsid proteins of WSSV.

Yahel G, Eerkes-Medrano DI, Leys SP (2006) Size independent selective filtration of ultraplankton by hexactinellid glass sponges. Aquatic Microbial Ecology 45 :181-194

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Selective feeding by flagellate and ciliate protists is important in shaping microbial communities in pelagic habitats. While less is known of predation on microbial communities in benthic habitats, the abundance and high filtering capacity of sponges suggests that they are key grazers. We studied the feeding preferences of two of the most common glass sponges of North-East Pacific fjords, Rhabdocalyptus dawsoni and Aphrocallistes vastus. Sponges were maintained in large darkened tanks supplied with running seawater from the nearby fjord. The water inhaled and exhaled by the sponges was simultaneously sampled and analyzed using a flow cytometer. Both sponges showed a similar (but not identical) feeding pattern, efficiently removing up to 99% of the most abundant bacterial cells, whereas clays, silt, and ’debris’ particles were expelled into the exhaled water. Filtration efficiencies were maximal for the relatively large and rare eukaryotic algae (3 to 5 pm, 86 +/- 9%) and for small non-photosynthetic bacteria (< 0.4 mu m, 89 +/- 10%), while intermediate sized non-photosynthetic bacteria characterized by higher nucleic acid content were efficiently removed in February (92 +/- 3%) when overall plankton concentration was low, but not in July (28 +/- 16%). The intermediate sized photosynthetic prokaryote Synechococcus (1.1 to 1.5 mu m) was also less preferred. Detailed analysis of the ultrastructure of the glass sponge filtration apparatus argues against possible ’by-pdss’ routes. We suggest that selective filtration involves individual processing, recognition, sorting, and transport of each particle through the sponges’ syncytial tissue. Selective grazing by glass sponges, like their pelagic protozoan counterparts, could be an important mechanism shaping microbial communities in the detrital food web of North-East Pacific fjords.

Yang H, Guo X (2006) Tetraploid induction by inhibiting mitosis I with heat shock, cold shock, and nocodazole in the hard clam Mercenaria mercenaria (Linnaeus, 1758). Mar Biotechnol (NY) 8 :501-510


Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38 degrees C), cold shock (1, 4, and 7 degrees C), and nocodazole (0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23 degrees C, and lasted for 10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae and juveniles was determined with flow cytometry. Heat shock of 35 and 38 degrees C was effective in inhibiting mitosis I, producing 54% to 89% tetraploid larvae. Heat shock of 32 degrees C accelerated embryonic development without inhibiting mitosis or producing tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids in the 4 degrees C and 7 degrees C treatment groups. Cold shock of 1 degrees C produced 31% tetraploid larvae in one replicate, with none surviving to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids. This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none of the induced tetraploids survived to juvenile stage.

Zamora-Ley IM, Gardinali PR, Jochem FJ (2006) Assessing the effects of Irgarol 1051 on marine phytoplankton populations in Key Largo Harbor, Florida. Mar Pollut Bull 52 :935-941


The antifouling boosting agent Irgarol 1051 is a strong inhibitor of the photosystem II (PSII) with high efficiency/toxicity towards algae. However, because some phytoplankton species are more sensitive to Irgarol than others, its persistent release into the environment could result in adverse changes in the phytoplankton community structure at heavily impacted sites such as marinas. Continuous monitoring in the Florida Keys showed Irgarol concentrations of up to 635 ngL(-1) in the canal system leading to Key Largo Harbor Marina (KLH) with a sharp decrease in concentration at stations offshore from the mouth of the canal. Preliminary phytoplankton community assessments from surface water samples collected in KLH between February and August 2004 showed changes in several phytoplankton species in concordance with the increase of the herbicide concentrations. Typical responses include an increase in the abundance of eukaryotes and Cryptomonas sp. as Irgarol concentrations increase.

Zhou GZ, Li ZQ, Zhang QY (2006) Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus. Viral Immunology 19 :637-645

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Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.