2008

mardi 21 avril 2009
par   G. Grégori

Abboudi M, Surget SM, Rontani JF, Sempere R, Joux F (2008) Physiological alteration of the marine bacterium Vibrio angustum S14 exposed to simulated sunlight during growth. Curr Microbiol 57 :412-417

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18769855

Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator : visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.


Ahn G, Hwang I, Park E, Kim J, Jeon YJ, Lee J, Park JW, Jee Y (2008) Immunomodulatory effects of an enzymatic extract from Ecklonia cava on murine splenocytes. Mar Biotechnol (NY) 10 :278-289

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18172723

We investigated whether the brown seaweed Alariaceae Ecklonia cava (E. cava) has immunological effects on splenocytes in vitro. For that purpose, we prepared an enzymatic extract from E. cava (ECK) by using the protease, Kojizyme. Here, ECK administered to ICR mice dramatically enhanced the proliferation of their splenocytes and increased the number of their lymphocytes, monocytes and granulocytes. In flow cytometry assays performed to identify in detail the specific phenotypes of these proliferating cells after ECK treatment, the numbers of CD4(+) T cells, CD8(+) T cells and CD45R/B220(+) B cells increased significantly compared to those in untreated controls. In addition, the mRNA expression and production level of Th1-type cytokines, i.e., TNF-alpha and IFN-gamma, were down-regulated, whereas those of Th2-type cytokines, i.e., IL-4 and IL-10, were up-regulated by ECK. Overall, this dramatic increase in numbers of splenocytes indicated that ECK could induce these cells to proliferate and could regulate the production of Th1- as well as Th2-type cytokines in immune cells. These results suggest that ECK has the immunomodulatory ability to activate the anti-inflammatory response and/or suppress the proinflammatory response, thereby endorsing its usefulness as therapy for diseases of the immune system.


Barbee GC, Barich J, Duncan B, Bickham JW, Matson CW, Hintze CJ, Autenrieth RL, Zhou GD, McDonald TJ, Cizmas L, Norton D, Donnelly KC (2008) In situ biomonitoring of PAH-contaminated sediments using juvenile coho salmon (Oncorhynchus kisutch). Ecotoxicol Environ Saf 71 :454-464

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18304636

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous marine and freshwater sediment contaminants. Extensive data exist to confirm that PAHs are toxic to aquatic receptors. However, limited information is available regarding the bioavailability and genotoxicity of sediment PAHs to aquatic organisms. This study investigated an integrated biomonitoring approach using chemical analyses and biomarkers to characterize the bioavailability and genotoxicity of a complex PAH mixture in freshwater lake sediments associated with a former manufactured gas plant (MGP). Sediment PAH genotoxicity was assessed by flow cytometry (FCM), DNA adduct (32)P-postlabeling, and erythrocyte micronuclei in juvenile coho salmon (Oncorhynchus kisutch) caged in the water column. Significant PAH-induced genotoxicity was observed with FCM and (32)P-postlabeling, but not with erythrocyte micronuclei. Chromosome damage in peripheral blood and hepatic DNA adducts correlated with sediment, but not water column PAH concentrations. Total hepatic DNA adducts in salmon caged nearest the former MGP facility was 39+/-6.5 (RALx10(9)), while salmon caged in a reference lake had 28+/-2.3 total hepatic DNA adducts per 10(9) nucleotides. These results indicate that in situ biomonitoring using biomarkers and caged fish can be a sensitive indicator of genotoxic PAHs in sediments.


Belzile C, Brugel S, Nozais C, Gratton Y, Demers S (2008) Variations of the abundance and nucleic acid content of heterotrophic bacteria in Beaufort Shelf waters during winter and spring. Journal of Marine Systems 74 :946-956

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Depth profiles of heterotrophic bacteria abundance were measured weekly over a 6-month period from December to May in Franklin Bay, a 230 m-deep coastal Arctic Ocean site of the southeastern Beaufort Sea. Total bacteria, low nucleic acid (LNA) and high nucleic acid (HNA) bacteria abundances were measured using (low cytometry after SYBR Green 1 staining. The HNA bacteria abundance in surface waters started to increase 5-6 weeks after phytoplankton growth resumed in spring, increasing from 1 x 10(5) to 3 x 10(5) cells mL(-1) over an 8-week period, with a net growth rate of 0.018 d(-1). LNA bacteria response was delayed by more than two months relative to the beginning of the phytoplankton biomass accumulation and had a lower net growth rate of 0.013 d(-1). The marked increase in bacterial abundance occurred before any significant increase in organic matter input from river discharge (as indicated by the unchanged surface water salinity and DOC concentrations), and in the absence of water temperature increase. The abundance of bacteria below the halocline was relatively high until January (up to 5 x 10(5) cells mL(-1)) but then decreased to values close to 2 x 10(5) cells mL(-1). The three-fold bacterial abundance increase observed in surface waters in spring was mostly due to HNA bacteria, supporting the idea that these cells are the most active. (C) 2007 Elsevier B.V. All rights reserved.


Berney M, Vital M, Hulshoff I, Weilenmann HU, Egli T, Hammes F (2008) Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Res 42 :4010-4018

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Fast and accurate monitoring of chemical and microbiological parameters in drinking water is essential to safeguard the consumer and to improve the understanding of treatment and distribution systems. However, most water utilities and drinking water guidelines still rely solely on time-requiring heterotrophic plate counts (HPC) and plating for faecal indicator bacteria as regular microbiological control parameters. The recent development of relative simple bench-top flow cytometers has made rapid and quantitative analysis of cultivation-independent microbial parameters more feasible than ever before. Here we present a study using a combination of cultivation-independent methods including fluorescence staining (for membrane integrity, membrane potential and esterase activity) combined with flow cytometry and total adenosine tri-phosphate (ATP) measurements, to assess microbial viability in drinking water. We have applied the methods to different drinking water samples including non-chlorinated household tap water, untreated natural spring water, and commercially available bottled water. We conclude that the esterase-positive cell fraction, the total ATP values and the high nucleic acid (HNA) bacterial fraction (from SYBR Green I staining) were most representative of the active/viable population in all of the water samples. These rapid methods present an alternative way to assess the general microbial quality of drinking water as well as specific events that can occur during treatment and distribution, with equal application possibilities in research and routine analysis.


Blanco GA, Malchiodi EL, De Marzi MC (2008) Cellular clot formation in a sipunculan worm : entrapment of foreign particles, cell death and identification of a PGRP-related protein. J Invertebr Pathol 99 :156-165

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Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.


Bonilla-Findji O, Malits A, Lefevre D, Rochelle-Newall E, Lemee R, Weinbauer MG, Gattuso JP (2008) Viral effects on bacterial respiration, production and growth efficiency : Consistent trends in the Southern Ocean and the Mediterranean Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography 55 :790-800

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To investigate the potential effects of viruses on bacterial respiration (BR), production (BP) and growth efficiency (BGE), experiments were performed using natural microbial communities from the coastal Mediterranean Sea, from a typical high-nutrient low-chlorophyll (HNLC) region in the Southern Ocean and from a naturally iron (Fe)-fertilized algal bloom above the Kerguelen Plateau (Southern Ocean). Seawater was sequentially filtered and concentrated to produce a bacterial concentrate, a viral concentrate and a virus-free ultrafiltrate. The combination of all three fractions served as treatments with active viruses. Heating or microwaving was used to inactivate viruses for the control treatments. Despite the differences in the initial trophic state and community composition of the study sites, consistent trends were found. In the presence of active viruses, BR was stimulated (up to 113%), whereas BP and BGE were reduced (up to 51%). Our results suggest that viruses enhance the role of bacteria as oxidizers of organic matter, hence as producers of CO2, and remineralizers of CO2, N, P and Fe. In the context of Fe-fertilization, this has important implications for the final fate of organic carbon in marine systems. (C) 2008 Elsevier Ltd. All rights reserved.


Brunet C, Casotti R, Vantrepotte V (2008) Phytoplankton diel and vertical variability in photobiological responses at a coastal station in the Mediterranean Sea. Journal of Plankton Research 30 :645-654

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Photophysiological parameters provide useful insights into the effects of environmental forcings on phytoplankton physiology. We present data on the short-term photoacclimative responses of phytoplankton throughout the water column during a diel sampling (every 1.5-3 h for 33 h) at a coastal station in the Gulf of Naples (Italy) in November 1996. Liposoluble pigments (high performance liquid chromatography), variable fluorescence (Prim-Prod probe) and picoplankton cell counts and autofluorescence (flow cytometry) were investigated every 1.5-3 h over a period of 33 h. The phytoplankton was phased to the alternation of light and dark and also showed acclimation to the different light intensities. Photoprotective pigments were synthesized during the day at the surface (0-20 m) and were significantly correlated with light intensity changes, as well as with the effective quantum yield of fluorescence. At night, recovery of photosystems from excess light was observed as was redistribution of nutrients and algae due to vertical convective motions caused by thermal dissipation. Equations linking photobiological parameters and time or light evolution were inferred to obtain kinetic coefficients. These were then used as biological tracers of vertical mixing whose velocity in the surface layer was estimated to be < 0.05 cm s(-1).


Cai H, Jiao N (2008) Diversity and abundance of nitrate assimilation genes in the northern South china sea. Microb Ecol 56 :751-764

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Marine heterotrophic microorganisms that assimilate nitrate play an important role in nitrogen and carbon cycling in the water column. The nasA gene, encoding the nitrate assimilation enzyme, was selected as a functional marker to examine the nitrate assimilation community in the South China Sea (SCS). PCR amplification, restriction fragment length polymorphism (RFLP) screening, and phylogenetic analysis of nasA gene sequences were performed to characterize in situ nitrate assimilatory bacteria. Furthermore, the effects of nutrients and other environmental factors on the genetic heterogeneity of nasA fragments from the SCS were evaluated at the surface in three stations, and at two other depths in one of these stations. The diversity indices and rarefaction curves indicated that the nasA gene was more diverse in offshore waters than in the Pearl River estuary. The phylotype rank abundance curve showed an abundant and unique RFLP pattern in all five libraries, indicating that a high diversity but low abundance of nasA existed in the study areas. Phylogenetic analysis of environmental nasA gene sequences further revealed that the nasA gene fragments came from several common aquatic microbial groups, including the Proteobacteria, Cytophaga-Flavobacteria (CF), and Cyanobacteria. In addition to the direct PCR/sequence analysis of environmental samples, we also cultured a number of nitrate assimilatory bacteria isolated from the field. Comparison of nasA genes from these isolates and from the field samples indicated the existence of horizontal nasA gene transfer. Application of real-time quantitative PCR to these nasA genes revealed a great variation in their abundance at different investigation sites and water depths.


Canesi L, Ciacci C, Betti M, Fabbri R, Canonico B, Fantinati A, Marcomini A, Pojana G (2008) Immunotoxicity of carbon black nanoparticles to blue mussel hemocytes. Environ Int 34 :1114-1119

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The potential for human and ecological toxicity associated with nanomaterials is a growing area of investigation. In mammalian cells, nanoparticles have been shown to induce inflammation and oxidative stress, and changes in cell signalling and gene expression. As the nanotechnology industries increase production, nanoscale products and by products will enter the aquatic environment, posing a possible threat to aquatic organisms. In particular, filter-feeding organisms may represent a unique target group for nanoparticle toxicology. In this work, the effects of commercial nanosized carbon black (NCB) on the immune cells, the hemocytes, of the bivalve mollusc Mytilus, and the possible mechanisms involved were investigated. The results demonstrate that NCB (1, 5, and 10 microg/ml), did not induce significant lysosomal membrane destabilization, as evaluated by the NR retention time assay. A concentration-dependent uptake of NCB by hemocytes was observed and it was associated by a rapid increase in extracellular lysozyme release, extracellular oxyradical production, and nitric oxide (NO) release. Moreover, at the highest concentration tested, NCB induced significant changes in mitochondrial parameters (decrease mitochondrial mass/number and membrane potential), as evaluated by flow cytometry. The effects of NCB were mediated by rapid activation of the stress-activated MAPKs (Mitogen Activated Protein Kinases) p38 and JNKs, that play a key role in immune and inflammatory responses. The results demonstrate that in mussel hemocytes like in mammalian cells NCB exposure can induce inflammatory processes, and indicate that bivalve immunocytes can represent a suitable model for investigating the effects and modes of action of nanoparticles in the cells of aquatic invertebrates.


Canesi L, Ciacci C, Betti M, Fabbri R, Canonico B, Fantinati A, Marcornini A, Pojana G (2008) Immunotoxicity of carbon black nanoparticles to blue mussel hemocytes. Environment International 34 :1114-1119

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The potential for human and ecological toxicity associated with nanomaterials is a growing area of investigation. In mammalian cells, nanoparticles have been shown to induce inflammation and oxidative stress, and changes in cell signalling and gene expression. As the nanotechnology industries increase production, nanoscale products and by products will enter the aquatic environment, posing a possible threat to aquatic organisms. In particular, filter-feeding organisms may represent a unique target group for nanoparticle toxicology. In this work, the effects of commercial nanosized carbon black (NCB) on the immune cells, the hemocytes, of the bivalve mollusc Mytilus, and the possible mechanisms involved were investigated. The results demonstrate that NCB (1, 5, and 10 mu g/ml), did not induce significant lysosomal membrane destabilization, as evaluated by the NR retention time assay. A concentration-dependent uptake of NCB by hemocytes was observed and it was associated by a rapid increase in extracellular lysozyme release, extracellular oxyradical production, and nitric oxide (NO) release. Moreover, at the highest concentration tested, NCB induced significant changes in mitochondrial parameters (decrease mitochondrial mass/number and membrane potential), as evaluated by flow cytometry. The effects of NCB were mediated by rapid activation of the stress-activated MAPKs (Mitogen Activated Protein Kinases) p38 and JNKs, that play a key role in immune and inflammatory responses. The results demonstrate that in mussel hemocytes like in mammalian cells NCB exposure can induce inflammatory processes, and indicate that bivalve immunocytes can represent a suitable model for investigating the effects and modes of action of nanoparticles in the cells of aquatic invertebrates. (c) 2008 Elsevier Ltd. All rights reserved.


Caruso G, Zappala G, Maimone G, Azzaro F, Raffa F, Caruso R (2008) Assessment of the abundance of actively respiring cells and dead cells within the total bacterioplankton of the Strait of Messina waters. Environmental Problems in Coastal Regions Vii 99 :15-24
232

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In aquatic ecosystems, the determination of the different physiological states coexisting within the bacterial assemblage is of great significance, as it may provide information on the effective role played by the viable component in the ecological processes. In May 2007, during a ship survey of the Messina Strait, a highly hydrodynamic system, an investigation was undertaken to study the abundance and distribution of the actively respiring and dead components of the bacterioplankton community. A dual-labelling procedure was applied, using SyBR Green as a probe of the total bacterioplankton community, in combination with the viability stains cyanotetrazolium chloride (CTC) or propidium iodide (PI), selective markers of actively respiring or membrane-damaged bacterial cells, respectively. Surface water samples were analysed onboard by a flow cytometer (FCM) and FCM counts were compared to the microscopic ones, obtained with the epifluorescence (EPI) method further performed in the laboratory. The study also pointed out the feasibility of the FCM approach as a rapid tool allowing the identification of the viable component and its discrimination from the dead or damaged one, also providing quantitative estimates correlated significantly with microscopic counts.


Casotti R (2008) Flow cytometry and marine monitoring for the early warning of biological risks. Cytometry Part A 73A :55-55

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Clementino MM, Vieira RP, Cardoso AM, Nascimento APA, Silveira CB, Riva TC, Gonzalez ASM, Paranhos R, Albano RM, Ventosa A, Martins OB (2008) Prokaryotic diversity in one of the largest hypersaline coastal lagoons in the world. Extremophiles 12 :595-604

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Araruama Lagoon is an environment characterized by high salt concentrations. The low raining and high evaporation rates in this region favored the development of many salty ponds around the lagoon. In order to reveal the microbial composition of this system, we performed a 16S rRNA gene survey. Among archaea, most clones were related to uncultured environmental Euryarchaeota. In lagoon water, we found some clones related to Methanomicrobia and Methanothermococcus groups, while in the saline pond water members related to the genus Haloarcula were detected. Bacterial community was dominated by clones related to Gamma-proteobacteria, Actinobacteria, and Synechococcus in lagoon water, while Salinibacter ruber relatives dominated in saline pond. We also detected the presence of Alpha-proteobacteria, Pseudomonas-like bacteria and Verrucomicrobia. Only representatives of the genus Ralstonia were cosmopolitan, being observed in both systems. The detection of a substantial number of clones related to uncultured archaea and bacteria suggest that the hypersaline waters of Araruama harbor a pool of novel prokaryotic phylotypes, distinct from those observed in other similar systems. We also observed clones related to halophilic genera of cyanobacteria that are specific for each habitat studied. Additionally, two bacterioplankton molecular markers with ecological relevance were analyzed, one is linked to nitrogen fixation (nifH) and the other is linked to carbon fixation by bacterial photosynthesis, the protochlorophyllide genes, revealing a specific genetic distribution in this ecosystem. This is the first study of the biogeography and community structure of microbial assemblages in Brazilian tropical hypersaline environments. This work is directed towards a better understanding of the free-living prokaryotic diversity adapted to life in hypersaline waters.


Connolly C (2008) Nanosensor developments in some European universities. Sensor Review 28 :18-21

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Purpose - This paper sets out to highlight selected projects in nanosensor research, demystify the technology and show potential applications in engineering fields.
Design/methodology/approach - Nano devices for sensing humidity and oxygen concentration are presented with applications in industrial monitoring. Then two approaches to the development of high-density optical memory are given. Next, a miniature flow cytometry system is described for the identification of marine micro-organisms and bacteria. Finally, photonic crystal structures with the ability to control and manipulate light are addressed.
Findings - "Nano" is currently a popular term, with a mass of publications in this area. Many universities have set up specialised centres for nanotechnology research. Crystalline materials with shape-selective nanopores can be designed to detect particular chemicals. Successful nanosensors are sensitive, simple, fast and low-cost.
Originality/value - This paper helps the general engineer to appreciate some aspects of nanotechnology. References to recent publications allow engineers to follow up their interests.


Cunha C, Doadrio I, Coelho MM (2008) Speciation towards tetraploidization after intermediate processes of non-sexual reproduction. Philosophical Transactions of the Royal Society B-Biological Sciences 363 :2921-2929

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Polyploidy, hybridization and variation in mating systems are central issues for a deeper understanding of animal evolution. The Iberian species Squalius alburnoides represents an example combining all three phenomena. Previous studies showed that S. alburnoides populations are mainly composed of triploid and diploid hybrid forms ( mainly females), and that the tetraploid forms are rare or absent. Both populations from the Douro drainage reveal a distinct scenario : tetraploid individuals represent 85.6 - 97.5% of the population, with no sex ratio bias observed. Based on the flow cytometry measurements of blood and spermatozoa cells, microsatellite loci and experimental crosses, we describe here, for the first time, two symmetric allotetraploid populations ( CCAA) that resumed normal meiosis after undergoing intermediate processes of non- sexual reproduction to give rise to a new sexually reproducing polyploid species. Prezygotic ( habitat selection and assortative mating) and postzygotic mechanisms ( nonviable embryos) are responsible for the reproductive isolation from other forms of the S. alburnoides complex ( e. g. CA, CAA). This example illustrates how hybrid polyploid complexes may lead to speciation.


Czechowska K, Johnson DR, van der Meer JR (2008) Use of flow cytometric methods for single-cell analysis in environmental microbiology. Curr Opin Microbiol 11 :205-212

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18562243

Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays.


da Silva TL, Reis A (2008) The use of multi-parameter flow cytometry to study the impact of n-dodecane additions to marine dinoflagellate microalga Crypthecodinium cohnii batch fermentations and DHA production. Journal of Industrial Microbiology & Biotechnology 35 :875-887

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The physiological response of Crypthecodinium cohnii batch cultivations and docosahexaenoic acid (DHA) production to n-dodecane additions were studied. Different n-dodecane concentrations [0, 0.5, 1, 2.5, 5, 10 and 20% (v/v)] were added to preliminary shake flask cultivations. The n-dodecane fraction that gave best results in terms of biomass and DHA production was 0.5% (v/v). The n-dodecane fractions of 2.5, 5, 10 and 20% (v/v) to C. cohnii preliminary shake flask cultures inhibited the microalgal growth and DHA production, although a high proportion of cells with intact cytoplasmic membrane was present in the end of these fermentations. After the addition of a pulse of n-dodecane (0.5% v/v) to C. cohnii exponential growing cells in a bioreactor, glucose uptake volumetric rate increased 2.5-fold, while biomass production volumetric rate increased 2.8-fold. The specific growth rate was increased 1.5-fold. The DHA % in biomass, DHA % of TFA and DHA concentration also increased (54, 22 and 58%, respectively), after the n-dodecane addition. At this n-dodecane fraction (0.5% v/v), multi-parameter flow cytometry demonstrated that C. cohnii cell membrane integrity was not affected. The results demonstrated that the addition of 0.5% of n-dodecane (v/v) to C. cohnii fermentations can be an easy and cheap way for enhancing the biomass and DHA production, avoiding the use of high speed rates (resulting in important power agitation costs) that affects the microalga proliferation and increases the bioprocess costs. A new strategy to improve the DHA production from this microalga in two-phase large-scale bioreactors is now in progress.


Debelius B, Forja JM, Del Valls A, Lubian LM (2008) Effect of linear alkylbenzene sulfonate (LAS) and atrazine on marine microalgae. Marine Pollution Bulletin 57 :559-568

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Five marine microalgae (Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana), in the same biovolume quantity, were exposed to 72 h growth-inhibition tests with atrazine and LAS. In all cases, the inhibition effect of atrazine was higher than that of LAS up to two orders of magnitude higher in the case of T chuii. In a second part of the study, initial cellular densities for each microalga strain and fixed organic compound concentration were varied, and results show density has a clear influence in growth inhibition tests. Finally, the sum of all data obtained in the study was expressed in terms of "toxic cellular quota" (mass of chemical substance per cell). The result was a sigmoid curve with a good fit, including the two main factors in toxicity tests, initial cellular density and contaminant concentration. This toxic cellular quota exhibits a general tendency to increase with cell volume/size. (c) 2008 Elsevier Ltd. All rights reserved.


Diaz MR, Dunbar SA, Jacobson JW (2008) Multiplexed detection of fungal nucleic acid signatures. Curr Protoc Cytom Chapter 13 :Unit13 19

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Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high-throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies.


Doolittle DF, Li WKW, Wood AM (2008) Wintertime abundance of picoplankton in the Atlantic sector of the Southern Ocean. Nova Hedwigia :147-160

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Data on the distribution of picophytoplankton from the Southern Ocean are relatively scant and primarily collected during the austral spring and summer. During the ICEFISH (International Collaborative Expedition to collect and study Fish Indigenous to Sub-Antarctic Habitats) expedition conducted in the austral winter, 2004, we examined the abundance of picophytoplankton in surface waters along a 366 km W-E transect at similar to 55 degrees S latitude between the South Sandwich Islands and Bouvetoya Island, a 2780 kin S-N transect from Bouvetoya Island to Tristan da Cunha Island, and a 2050 kin W-E transect extending east from Tristan da Cunha toward Capetown, South Africa. The cruise track traversed a region of the Antarctic Circumpolar Current (ACC) that included four major frontal features : the Southern Antarctic Circumpolar Current Front (SACCF), the Antarctic Polar Front (APF), the Subantarctic Front (SAF), and the Subtropical Front (STF). In waters less than 1 degrees C, and south of the SACCF, picoeukaryotes represented more than 99% of the picophytoplankton in the community. Phycoerythrin-containing picoplankton 2 mu m in equivalent spherical diameter (ESD) were observed along our S-N transect once water temperatures exceeded 1.3 degrees C, placing their southernmost limit of distribution close to the Antarctic Polar Front. Substantial populations of these organisms, which had a flow cytometric signature comparable to those of PE-containing marine Synechococcus and other PE-containing picocyanobacteria, were seen in all samples collected in water >4 degrees C and north of the APF. Thus, both PE-containing picoplankton <2 mu m in equivalent spherical diameter (ESD) and picoeukaryotes appear to be part of the phytoplankton community in Antarctic polar waters year-round. In contrast, Prochlorococcus did not appear in water <10 degrees C or south of the southern expression of the STF, as expected from other reports. A strong linear relationship exists between log phytoplankton abundance and temperature, which is only observed when all functional groups are pooled. Picoplankton distributions show marked changes at frontal boundaries and support the hypothesis that water mass related phenomena, and not just temperature alone, determine phytoplankton community structure in the Southern Ocean.


Duchemin MB, Auffret M, Wessel N, Fortier M, Morin Y, Pellerin J, Fournier M (2008) Multiple experimental approaches of immunotoxic effects of mercury chloride in the blue mussel, Mytilus edulis, through in vivo, in tubo and in vitro exposures. Environmental Pollution 153 :416-423

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Biological impairments due to mercury discharge into the environment are now an issue of global concern. From the three forms of mercury found in aquatic ecosystems, the immunotoxic effects of mercury chloride were examined in the model animal, the blue mussel. In order to investigate the toxic potency of this chemical, three exposure regimes were carried out : chronic exposure of groups of individuals, a new protocol "in tubo" designed for sub-acute exposures of individuals, and acute exposures of target cells. Chronic exposure revealed significant immunotoxic effects after 7 days at 10(-6) M, while acute exposures showed significant inhibition of phagocytosis at 10(-4) M and 10(-3) M. In sub-acute exposures both circulating haemocytes and haemocyte mortality increased at 10(-4) M and 10(-3) M while phagocytosis and the clearance rate drew hormetic toxic effects on healthy individuals. These results suggest the use of the "in tubo" design for bivalve toxicological individual studies. (C) 2007 Elsevier Ltd. All rights reserved.


Duchemin MB, Wessel N, Fournier M, Auffret M (2008) Flow cytometric measurement of the clearance rate in the blue mussel Mytilus edulis and the development of a new individual exposure system for aquatic immunotoxicological studies. Environ Pollut 153 :492-496

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Animals in poor health condition are not relevant biological models. The current study focused on the use of the clearance rate of Mytilus edulis to assess the gross physiological condition of individuals maintained in stressful experimental conditions. This approach was developed in a new, highly controlled experimental exposure device designed to investigate individual responses in aquatic ecotoxicological studies. Both clearance rate values and immune parameters analysis indicated that the health condition of mussels kept in 50ml tubes for 24h or 48h was not altered compared to controls, while most parameters were depressed after 72h. Moreover, this study confirms the relevance of flow cytometric for the measurement of clearance rate compared to techniques utilizing microscopy. Current results prompted us to perform further 24h chemical exposure using this "in tubo" device.


Duhamel S, Gregori G, Van Wambeke F, Mauriac R, Nedoma J (2008) A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry. J Microbiol Methods 75 :269-278

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18639593

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g ; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4’,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.


Engel A, Schulz KG, Riebesell U, Bellerby R, Delille B, Schartau M (2008) Effects of CO2 on particle size distribution and phytoplankton abundance during a mesocosm bloom experiment (PeECE II). Biogeosciences 5 :509-521

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The influence of seawater carbon dioxide (CO2) concentration on the size distribution of suspended particles (2-60 mu m) and on phytoplankton abundance was investigated during a mesocosm experiment at the large scale facility (LFS) in Bergen, Norway, in the frame of the Pelagic Ecosystem CO2 Enrichment study (PeECE II). In nine outdoor enclosures the partial pressure of CO2 in seawater was modified by an aeration system to simulate past (similar to 190 mu atm CO2), present (similar to 370 mu atm CO2) and future (similar to 700 mu atm CO2) CO2 conditions in triplicates. Due to the initial addition of inorganic nutrients, phytoplankton blooms developed in all mesocosms and were monitored over a period of 19 days. Seawater samples were collected daily for analysing the abundance of suspended particles and phytoplankton with the Coulter Counter and with Flow Cytometry, respectively. During the bloom period, the abundance of small particles (< 4 mu m) significantly increased at past, and decreased at future CO2 levels. At that time, a direct relationship between the total-surface-to-total-volume ratio of suspended particles and DIC concentration was determined for all mesocosms. Significant changes with respect to the CO2 treatment were also observed in the phytoplankton community structure. While some populations such as diatoms seemed to be insensitive to the CO2 treatment, others like Micromonas spp. increased with CO2, or showed maximum abundance at present day CO2 (i.e. Emiliania huxleyi). The strongest response to CO2 was observed in the abundance of small autotrophic nano-plankton that strongly increased during the bloom in the past CO2 mesocosms. Together, changes in particle size distribution and phytoplankton community indicate a complex interplay between the ability of the cells to physiologically respond to changes in CO2 and size selection. Size of cells is of general importance for a variety of processes in marine systems such as diffusion-limited uptake of substrates, resource allocation, predator-prey interaction, and gravitational settling. The observed changes in particle size distribution are therefore discussed with respect to biogeochemical cycling and ecosystem functioning.


Espinosa EP, Allam B, Ford SE (2008) Particle selection in the ribbed mussel Geukensia demissa and the Eastern oyster Crassostrea virginica : Effect of microalgae growth stage. Estuarine Coastal and Shelf Science 79 :1-6

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We studied particle selection in the ribbed mussel Geukensia demissa, an important suspension-feeding inhabitant of estuaries and intertidal zones of salt marshes along the Atlantic coast of North America. Adult mussels were fed on several mixtures of microalgal cultures (1) in exponential or (2) in stationary phase of growth, and the proportional occurrence of algal species in pseudofeces was examined by flow cytometry. The Eastern oyster, Crassostrea virginica, was chosen as a reference. Results showed that both mussels and oysters were able to selectively ingest or reject our experimental microalgae. Moreover, the pre-ingestive particle selection was affected by microalgal growth phase, particularly in mussels. For instance, the sorting efficiency index increased significantly in mussels fed with a blend made of Nitzschia closterium, Isochrysis sp. and Tetraselmis suesica harvested in stationary growth phase, as compared to the same blend made with microalgae in exponential growth phase. Isochrysis sp. and T suesica were preferentially ingested by both bivalves whereas N. closterium, was preferentially rejected in pseudofeces. These results demonstrate particle selection in ribbed mussel and underline the effect of algae growth phase on the sorting mechanisms. (C) 2008 Elsevier Ltd. All rights reserved.


Fafandel M, Bihari N, Smodlaka M, Ravlic S (2008) Hemocytes/coelomocytes DNA content in five marine invertebrates : cell cycles and genome sizes. Biologia 63 :730-736

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The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.


Falcioni T, Papa S, Gasol JM (2008) Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl Environ Microbiol 74 :1767-1779

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18223113

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Gregori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability : active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 microg ml(-1) of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.


Figueroa RI, Bravo I, Garces E (2008) The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum. Harmful Algae 7 :653-663

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Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced : resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (< 5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1-6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 +/- 3.0 mu m) had a dormancy period of 1-3 months, whereas temporary asexual cysts (32.5 +/- 5.4 mu m) germinated in less than 7 days. (c) 2008 Elsevier B.V. All rights reserved.


Frada M, Probert I, Allen MJ, Wilson WH, de Vargas C (2008) The "Cheshire Cat" escape strategy of the coccolithophore Emiliania huxleyi in response to viral infection. Proceedings of the National Academy of Sciences of the United States of America 105 :15944-15949

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The coccolithophore Emiliania huxleyi is one of the most successful eukaryotes in modern oceans. The two phases in its haplodiploid life cycle exhibit radically different phenotypes. The diploid calcified phase forms extensive blooms, which profoundly impact global biogeochemical equilibria. By contrast, the ecological role of the noncalcified haploid phase has been completely overlooked. Giant phycodnaviruses (Emiliania huxleyi viruses, EhVs) have been shown to infect and lyse diploid-phase cells and to be heavily implicated in the regulation of populations and the termination of blooms. Here, we demonstrate that the haploid phase of E. huxleyi is unrecognizable and therefore resistant to EhVs that kill the diploid phase. We further show that exposure of diploid E. huxleyi to EhVs induces transition to the haploid phase. Thus we have clearly demonstrated a drastic difference in viral susceptibility between life cycle stages with different ploidy levels in a unicellular eukaryote. Resistance of the haploid phase of E. huxleyi provides an escape mechanism that involves separation of meiosis from sexual fusion in time, thus ensuring that genes of dominant diploid clones are passed on to the next generation in a virus-free environment. These "Cheshire Cat" ecological dynamics release host evolution from pathogen pressure and thus can be seen as an opposite force to a classic "Red Queen" coevolutionary arms race. In E. huxleyi, this phenomenon can account for the fact that the selective balance is tilted toward the boom-and-bust scenario of optimization of both growth rates of calcifying E. huxleyi cells and infectivity of EhVs.


Fujiwara K, Suematsu H, Kiyomiya E, Aoki M, Sato M, Moritoki N (2008) Size-dependent toxicity of silica nano-particles to Chlorella kessleri. J Environ Sci Health A Tox Hazard Subst Environ Eng 43 :1167-1173

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18584432

SiO(2) nano-particles were found to exhibit size-dependent toxicity toward the alga, Chlorella kessleri. Small SiO(2) nano-particles exhibit stronger toxicity : 50% inhibitory concentrations (IC(50)) value for 5 nm = 0.8 +/- 0.6%, 26 nm = 7.1 +/- 2.8%, and 78 nm = 9.1 +/- 4.7%. Enlargement of the cell body was observed by flow cytometry, which is due to the presence of structures that obstructed cell division. Optical and transmission microscopes were used to observe coagulated cells with incomplete division. Although the physiological effect of SiO(2) nano-particles was not clear, SiO(2) nano-particles are toxic, at least for algae in aquatic media. Under the transmission electron microscope, several amorphous structures appeared in the cells that were exposed to 5-nm silica nano-particles.


Gagnaire B, Duchemin M, Auffret M, Thomas-Guyon H, Renault T (2008) Comparison of hemocyte parameters in the pericardial cavity and the adductor muscle sinus in the Pacific oyster, Crassostrea gigas using two types of flow cytometers. Aquatic Living Resources 21 :39-43

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Parameters of hemocyte populations have been considered as relevant indicators of bivalve health and are currently used in immunotoxicological studies. Hemocytes in hemolymph can be collected by puncturing either the pericardial cavity or the adductor muscle sinus with a syringe. Flow cytometry is a methodological approach that is increasingly being used in laboratories for the study of hemocyte parameters in aquatic invertebrates. However, various protocols for hemocyte processing in laboratories equipped with different types of cytometers have been published. In this context, two flow cytometers ( EPICS XL4 (R), Beckman Coulter and FacsCalibur (R), Becton Dickinson) and two sites of hemocyte collection ( pericardial cavity and adductor muscle sinus) were compared for the analysis of hemocyte parameters in the Pacific oyster, Crassostrea gigas. Hemolymph cells were analyzed in terms of their number and organelle contents. Cell mortality, phagocytosis, non specific esterase, extension of the lysosomal compartment and production of reactive oxygen species were quantified. The results showed that the phagocytic index was higher for hemocytes obtained in the muscle sinus hemolymph. The results are discussed with respect to the potential use of flow cytometry as a tool for hemocyte studies in bivalves.


Gendreau E, Romaniello S, Barad S, Leymarie J, Benech-Arnold R, Corbineau F (2008) Regulation of cell cycle activity in the embryo of barley seeds during germination as related to grain hydration. Journal of Experimental Botany 59 :203-212

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Various studies indicate that cell division is a post-germination phenomenon, with radicle protrusion occurring by cell elongation, while others demonstrate that induction of the cell cycle occurs in osmo-conditioned seeds prior to radicle growth. The aim of the present work was to investigate the occurrence of the cell cycle during germination as related to grain hydration, using : (i) a flow cytometry technique to estimate the percentage of cell nuclei in G(1) and G(2) phases of the cell cycle ; and (ii) reverse transcription-PCR (RT-PCR) in order to characterize the expression of the genes encoding cyclin-dependent kinases (CDKA1, CDKB1, and CDKD1) and cyclins (CYCA3, CYCB1, and CYCD4), the main genes involved in the cell cycle and its regulation. Radicle tips of embryos were isolated from seeds placed for various times on water at 30 degrees C and from grains partially hydrated at moisture contents ranging from 11% to 51% fresh weight (FW), which prevent radicle elongation. Abscisic acid (ABA) contents of the embryos during seed germination at 30 degrees C and after 48 h of partial hydration were also measured. In dry embryos, cells are mostly arrested in the G(1) phase of the cell cycle (82%), the remaining cells being in the G(2) phase, and the ABA content of the embryo was 432.7 ng g(-1) dry weight (DW). Seed imbibition was associated with a sharp decrease in ABA content as early as 5 h, while the cell cycle reactivation was a late process taking place ; similar to 4-6 h prior to radicle protrusion. Hydration of seeds resulted in a decrease in embryo ABA content, but it remained at a high level (207-273 ng g(-1) DW) evenafter 48 h at 0.41-0.51 g H2O g(-1) FW. The cell population of the radicle tips in the G(2) phase of the cell cycle, i.e. 4C nuclei, increased from 9% up to 34% at a moisture content of 51% FW. In dry seeds, CDKA1 and CDKD1 mRNAs were present at low levels, but transcripts of CDKB1, CYCA3, CYCB1, and CYCD4 were not detected. Radicle protrusion was associated with a higher expression of CDKA1, CDKB1, CYCA3, and CYCB1. Blockage of germination of partially hydrated grains resulted in a reduction in the expression of CDKA1 and CDKB1, and of CYCA3 and CYCB1, and in a reinforcement of that of CDKD1 and CYCD4. Patterns of gene expression show differential sensitivity of the genes studied to hydration of the grain. They will be discussed with regard to embryo ABA content and embryo sensitivity to ABA.


Gescher C, Metfies K, Frickenhaus S, Knefelkamp B, Wiltshire KH, Medlin LK (2008) Feasibility of assessing the community composition of prasinophytes at the Helgoland roads sampling site with a DNA microarray. Applied and Environmental Microbiology 74 :5305-5316

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The microalgal class Prasinophyceae (Chlorophyta) contains several picoeukaryotic species, which are known to be common in temperate and cold waters and have been observed to constitute major fractions of marine picoplankton. However, reliable detection and classification of prasinophytes are mainly hampered by their small size and few morphological markers. Consequently, very little is known about the abundance and ecology of the members of this class. In order to facilitate the assessment of the abundance of the Prasinophyceae, we have designed and evaluated an 18S rRNA gene-targeted oligonucleotide microarray consisting of 21 probes targeting different taxonomic levels of prasinophytes. The microarray contains both previously published probes from other hybridization methods and new probes, which were designed for novel prasinophyte groups. The evaluation of the probe set was done under stringent conditions with 18S PCR fragments from 20 unialgal reference cultures used as positive targets. This microarray has been applied to assess the community composition of prasinophytes at Helgoland, an island in the North Sea where time series data are collected and analyzed daily but only for the nano-and microplankton-size fractions. There is no identification of prasinophytes other than to record them numerically in the flagellate fraction. The samples were collected every 2 weeks between February 2004 and December 2006. The study here demonstrates the potential of DNA microarrays to be applied as a tool for quick general monitoring of this important picoplanktonic algal group.


Gillard J, Devos V, Huysman MJ, De Veylder L, D’Hondt S, Martens C, Vanormelingen P, Vannerum K, Sabbe K, Chepurnov VA, Inze D, Vuylsteke M, Vyverman W (2008) Physiological and transcriptomic evidence for a close coupling between chloroplast ontogeny and cell cycle progression in the pennate diatom Seminavis robusta. Plant Physiol 148 :1394-1411

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18820084

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.


Gillard J, Devos V, Huysman MJJ, De Veylder L, D’Hondt S, Martens C, Vanormelingen P, Vannerum K, Sabbe K, Chepurnov VA, Inze D, Vuylsteke M, Vyverman W (2008) Physiological and Transcriptomic Evidence for a Close Coupling between Chloroplast Ontogeny and Cell Cycle Progression in the Pennate Diatom Seminavis robusta. Plant Physiology 148 :1394-1411

<Go to ISI> ://000260719500018

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.


Goanvec C, Theron M, Lacoue-Labarthe T, Poirier E, Guyomarch J, Le-Floch S, Laroche J, Nonnotte L, Nonnotte G (2008) Flow cytometry for the evaluation of chromosomal damage in turbot Psetta maxima (L.) exposed to the dissolved fraction of heavy fuel oil in sea water : a comparison with classical biomarkers. Journal of Fish Biology 73 :395-413

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Turbot Psetta maxima were exposed 5 days to the dissolved fraction of fuel oil number 2, then decontaminated over 30 days in clean sea water. Biliary metabolites and ethoxyresorufin-O-deethylase (EROD) activity were evaluated during and after the contamination. These results were compared with chromosomal damage measured by flow cytometry (FCM). Erythrocyte nuclear abnormality, micronuclei and immaturity were also evaluated over the exposure period. Biliary metabolites and EROD analyses showed a clear and early response : biliary metabolites were detected from the first day of contamination to the 14th day of depuration, EROD activity increased during the contamination period reached a maximum 3 days after the beginning of the decontamination and decreased to the control value after 1 month of depuration. FCM showed a bimodal response : a first increase of coefficient of variation of blood cell DNA content was observed during the contamination and a second one started after 14 days of depuration and was maintained for at least 2 weeks. Erythrocyte morphology analysis showed a strong increase in nuclear abnormality during the contamination period. These results confirm previous work and show that in the context of marine accidental pollution by heavy fuel oil, the measurements of chromosomal damage by FCM allow the detection of a genotoxic response in fishes. (C) 2008 The Authors Journal compilation (C) 2008 The Fisheries Society of the British Isles.


Gomez-Baena G, Lopez-Lozano A, Gil-Martinez J, Lucena JM, Diez J, Candau P, Garcia-Fernandez JM (2008) Glucose uptake and its effect on gene expression in prochlorococcus. PLoS ONE 3 :e3416

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18941506

The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation) was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively) is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon cycle in nature. Besides, the ability of assimilating carbon molecules could provide additional hints to comprehend the ecological success of Prochlorococcus.


Greisberger S, Dokulil MT, Teubner K (2008) A comparison of phytoplankton size-fractions in Mondsee, an alpine lake in Austria : distribution, pigment composition and primary production rates. Aquatic Ecology 42 :379-389

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Production rates, abundance, chlorophyll a (Chl a) concentrations and pigment composition were measured for three size classes (< 2 mu m, 2-11 mu m and > 11 mu m) of phytoplankton from May to December 2000 in deep, mesotrophic, alpine lake Mondsee in Austria. The study focuses on differences among phytoplankton size fractions characterised by their surface area to volume ratio ([mm(2) l(-1) : mm(3)l(-1)]), pigment distribution patterns and photosynthetic rates. Particular attention was paid to autotrophic picophytoplankton (APP, fraction < 2 mu m) since this size fraction differed significantly from the two larger size fractions. Among the three fractions, APP showed the highest surface area to volume ratios and a high persistence in the pattern of lipophilic pigments between temporarily and spatially successive samples (about 80% similarity of pigment composition between samples over seasons and depths). The epilimnetic abundance of APP varied seasonally with an annual maximum of 180 x 10(3) cells ml(-1) in June (at 4-9 m). The minimum (October at 12 m) was more than an order of magnitude lower (4.9 x 10(3) ml(-1)). APP peaked during autumn and contributed between 24% and 42% to the total area-integrated Chl a (10-23 mg m(-2)) and between 16% and 58% to total area-integrated production (5-64 mg m(-2) h(-1)) throughout seasons.


Guthrie HD, Woods LC, Long JA, Welch GR (2008) Effects of osmolality on inner mitochondrial transmembrane potential and ATP content in spermatozoa recovered from the testes of striped bass (Morone saxatilis). Theriogenology 69 :1007-1012

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The objective was to determine the effects of osmolality on the energy status of testicular spermatozoa of striped bass incubated in a TRIS free base-NaCl medium (pH 8) adjusted to either 300 (T300) or 600 mOsm/kg (T600) with NaCl. High mitochondrial inner transmembrane potential (Delta Psi(m)) was assessed (flow cytometry) with the mitochondrial probe 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) and ATP was measured with a luciferin-luciferase assay. Spermatozoa maintained on ice were equally viable (> 95% for T300 and T600) for up to 80 min, whereas sperm viability in artificial fresh water (FW) at 27 mOsm/kg decreased (P < 0.05) to 67% after 5 min, with only 3.5% viability at 25 min. After 20 min of staining, more spermatozoa (P < 0.05) maintained a high Delta Psi(m) in T300 than in T600 (80 and 50%, respectively). Sperm JC-1 aggregate (J(agg)) fluorescence intensity was also greater (P < 0.05) in T300 than in T600 (10 and 5 channel number). The J(agg) fluorescence was a function of oxidative phosphorylation ; the percentage of cells containing J(agg) fluorescence decreased to 3% in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an uncoupler of cell respiration and oxidative phosphorylation. After incubation for 30 min in the absence of CCCP, sperm ATP concentration was greater (P < 0.05) in T300 than in T600 (2.0 vs. 0.2 pmol/10(6) cells), but was below delectability in the presence of CCCP in either medium. In conclusion, we developed a unique approach to assess the energetic status of striped bass spermatozoa during storage and after activation, and concluded that the effects of osmolality must be considered in the design of activating and storage extenders to maintain striped bass sperm motility, viability, and fertility in vitro. (c) 2008 Published by Elsevier Inc.


Hammes F, Berney M, Wang Y, Vital M, Koster O, Egli T (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res 42 :269-277

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17659762

There are significantly more microbial cells in drinking water than what can be cultured on synthetic growth media. Nonetheless, cultivation-based heterotrophic plate counts (HPCs) are used worldwide as a general microbial quality parameter in drinking water treatment and distribution. Total bacterial cell concentrations are normally not considered during drinking water treatment as a design, operative or legislative parameters. This is mainly because easy and rapid methods for quantification of total bacterial cell concentrations have, up to now, not been available. As a consequence, the existing lack of data does not allow demonstrating the practical value of this parameter. In this study, we have used fluorescence staining of microbial cells with the nucleic acid stain SYBR((R)) Green I together with quantitative flow cytometry (FCM) to analyse total cell concentrations in water samples from a drinking water pilot plant. The plant treats surface water (Lake Zurich) through sequential ozonation, granular active carbon (GAC) filtration and membrane ultrafiltration (UF). The data were compared with adenosine tri-phosphate (ATP) measurements and conventional HPCs performed on the same water samples. We demonstrated that the impact of all three major treatment steps on the microbiology in the system could accurately be described with total cell counting : (1) ozonation caused chemical destruction of the bacterial cells ; (2) GAC filtration facilitated significant regrowth of the microbial community ; and (3) membrane UF physically removed the bacterial cells from the water. FCM typically detected 1-2 log units more than HPC, while ATP measurements were prone to interference from extracellular ATP released during the ozonation step in the treatment train. We have shown that total cell concentration measured with FCM is a rapid, easy, sensitive and importantly, a descriptive parameter of several widely applied drinking water treatment processes.


Hammes F, Berney M, Wang YY, Vital M, Koster O, Egli T (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Research 42 :269-277

<Go to ISI> ://000253045900026

There are significantly more microbial cells in drinking water than what can be cultured on synthetic growth media. Nonetheless, cultivation-based heterotrophic plate counts (HPCs) are used worldwide as a general microbial quality parameter in drinking water treatment and distribution. Total bacterial cell concentrations are normally not considered during drinking water treatment as a design, operative or legislative parameters. This is mainly because easy and rapid methods for quantification of total bacterial cell concentrations have, up to now, not been available. As a consequence, the existing lack of data does not allow demonstrating the practical value of this parameter. In this study, we have used fluorescence staining of microbial cells with the nucleic acid stain SYBR(R) Green I together with quantitative flow cytometry (FCM) to analyse total cell concentrations in water samples from a drinking water pilot plant. The plant treats surface water (Lake Zilrich) through sequential ozonation, granular active carbon (GAC) filtration and membrane ultrafiltration (UF). The data were compared with adenosine tri-phosphate (ATP) measurements and conventional HPCs performed on the same water samples. We demonstrated that the impact of all three major treatment steps on the microbiology in the system could accurately be described with total cell counting : (1) ozonation caused chemical destruction of the bacterial cells ; (2) GAC filtration facilitated significant regrowth of the microbial community ; and (3) membrane UF physically removed the bacterial cells from the water. FCM typically detected 1-2 log units more than HPC, while ATP measurements were prone to interference from extracellular ATP released during the ozonation step in the treatment train. We have shown that total cell concentration measured with FCM is a rapid, easy, sensitive and importantly, a descriptive parameter of several widely applied drinking water treatment processes. (C) 2007 Elsevier Ltd. All rights reserved.


Hibi K, Ushio H, Fukuda H, Mitsubayashi K, Hayashi T, Ren H, Endo H (2008) Immunomagnetic separation using carbonyl iron powder and flow cytometry for rapid detection of Flavobacterium psychrophilum. Anal Bioanal Chem 391 :1147-1152

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18438648

Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a serious problem in the aquaculture industry worldwide. Several methods to prevent and treat cold water disease have been studied. Although detection at the early stage of F. psychrophilum infection is very important for the prevention and treatment of cold water disease, an effective detection method has not yet been developed. The use of flow cytometry (FCM) for the rapid determination of bacterial cell numbers with high sensitivity is beginning to attract attention. Immunomagnetic separation (IMS) has also been used to detect F. psychrophilum. The purpose of the present study was to develop a method to quickly determine the number of bacterial cells by combining the FCM and IMS methods. Because samples can be more effectively concentrated using smaller magnetic beads and stronger magnetism, we used carbonyl iron powder as the magnetic beads for the IMS. The detection level of F. psychrophilum using FCM combined with IMS was 5 orders lower than that using FCM without IMS. The values determined using FCM combined with IMS strongly correlated with those obtained using the colony-counting method, in the range of approximately 10-10(8) colony-forming units per milliliter. One FCM assay could be completed within 60 s and the total assay time, including sample preparation, was less than 2 h. The combined method of FCM with IMS developed in this study can be used reliably for the rapid detection of F. psychrophilum.


Hirose M, Katano T, Hayami Y, Kaneda A, Kohama T, Takeoka H, Nakano S (2008) Changes in the abundance and composition of picophytoplankton in relation to the occurrence of a Kyucho and a bottom intrusion in the Bungo Channel, Japan. Estuarine Coastal and Shelf Science 76 :293-303

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The Bungo Channel in southwestern Japan receives both warm, called Kyucho, and cold deep-water intrusions (bottom intrusion) from the Pacific Ocean. Abundances of Prochlorococcus, Synechococcus, and eukaryotic picophytoplankton were monitored from 18 July to 17 August 2001 to clarify whether advected picophytoplankton from the Pacific Ocean can grow in the channel or not. Synechococcus cells were further discriminated into low- and high-PUB types according to their fluorescence property in flow cytometry. From 18 to 25 July, the water temperature decreased by 3 degrees C at a 5-m depth at all stations, indicating the occurrence of a bottom intrusion. From 25 July to 4 August, a Kyucho occurred and the water temperature rapidly increased. From 4 to 17 August, a bottom intrusion and a Kyucho both occurred twice, although the intensities were smaller than those occurring until 4 August. From 18 to 30 July, the abundance of both Prochlorococcus and a high-PUB type of Synechococcus drastically decreased because of a bottom intrusion ; however, the abundances rapidly increased due to the advection by a Kyucho. These advected cells increased from 4 to 17 August in the channel and Kitanada Bay. Changes in the abundance of low-PUB type of Synechococcus and eukaryotic picophytoplankton were less noticeable than those in the abundance of Prochlorococcus and high-PUB type. The present study demonstrated that oceanic picophytoplankton advected by the Kyucho could grow in the channel. However, abundances of low-PUB type and eukaryotic picophytoplankton increased higher than those of Prochlorococcus and high-PUB type did. Thus, these oceanic phytoplankters will be excluded when Kyucho does not occur for a long time. The co-occurrence of various types of picophytoplankton found in the channel is probably achieved by both Kyucho event and their growth capability in the channel. (C) 2007 Elsevier Ltd. All rights reserved.


Holtzendorff J, Partensky F, Mella D, Lennon JF, Hess WR, Garczarek L (2008) Genome streamlining results in loss of robustness of the circadian clock in the marine cyanobacterium Prochlorococcus marinus PCC 9511. J Biol Rhythms 23 :187-199

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18487411

The core oscillator of the circadian clock in cyanobacteria consists of 3 proteins, KaiA, KaiB, and KaiC. All 3 have previously been shown to be essential for clock function. Accordingly, most cyanobacteria possess at least 1 copy of each kai gene. One exception is the marine genus Prochlorococcus, which we suggest here has suffered a stepwise deletion of the kaiA gene, together with significant genome streamlining. Nevertheless, natural Prochlorococcus populations and laboratory cultures are strongly synchronized by the alternation of day and night, displaying 24-h rhythms in DNA replication, with a temporal succession of G1, S, and G2-like cell cycle phases. Using quantitative real-time PCR, we show here that in Prochlorococcus marinus PCC 9511, the mRNA levels of the clock genes kaiB and kaiC, as well as a few other selected genes including psbA, also displayed marked diel variations when cultures were kept under a light-dark rhythm. However, both cell cycle and psbA gene expression rhythms damped very rapidly under continuous light. In the closely related Synechococcus sp. WH8102, which possesses all 3 kai genes, cell cycle rhythms persisted over several days, in agreement with established cyanobacterial models. These data indicate a correlation between the loss of kaiA and a loss of robustness in the endogenous oscillator of Prochlorococcus and raise questions about how a basic KaiBC system may function and through which mechanism the daily "lights-on" and "lights-off" signal could be mediated.


Hwang CY, Cho BC (2008) Effects of storage on the estimates of virus-mediated bacterial mortality based on observation of preserved seawater samples with TEM. Aquatic Microbial Ecology 52 :263-271

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There are reports in the literature that the long-term storage (e.g. > 30 d) of fixative-preserved seawater samples causes significant decreases in bacterial (BA) and viral abundances (VA). However, the effects of storage on the frequency of visibly infected bacteria (FVIB), and consequently on bacterial mortality due to viral lysis (BMVL), remain to be evaluated. First, to determine the variables that facilitate the prediction of the FVIB value at the time of storage (i.e. FVIBi), we considered a bacterial community composed of 2 groups (i.e. visibly infected bacteria and the others) and assumed an exponential decay relationship for the bacteria in each group during the storage of preserved samples. In the hypothetical model, the FVIBi could be well estimated in terms of BA at the time of storage, a decay rate of BA, and FVIBf and BA(f) (i.e. FVIB and BA measured at the end of storage, respectively). Further, we tested this idea by applying it to 7 seawater samples that were preserved with 2% glutaraldehyde (final conc.) and stored at different temperatures for ca. 30 d. For the 3 preserved coastal samples, considerably better estimates of BMVL were obtained by applying the theoretical consideration to estimate the FVIBi (the BMVL was estimated to be 89.9 to 118.7% of the BMVL at the time of storage [BMVLi], which was the value calculated with FVIBi than if FVIBf was used (the BMVL was estimated to be 45.5 to 89.9% of the BMVLi value). Interestingly, estimates of the BMVL obtained for the 4 preserved offshore samples were comparable to the FVIB values measured at 3 to 5 d after the start of the experiments, as in the case of preserved coastal samples in the present study, suggesting that it may be possible to estimate FVIBi values for preserved offshore samples. It is recommended that the above-mentioned variables be measured in order to reliably estimate the FVIB value at the time of preservation for fixative-amended and frozen seawater samples that may be stored for a long time, such as oceanic cruises.


Jessen A, Randall A, Reinhart D, Daly L (2008) Effectiveness and Kinetics of Ferrate as a Disinfectant for Ballast Water. Water Environment Research 80 :561-569

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This study examined whether ferrate could meet the international standards for successful ballast water treatment, including final concentrations of less than 1 CFU/mL of Enterococci, less than 2.5 CFU/mL of Escherichia coli, and less than 1 CFU/100 mL of Vibrio cholerae. Pure cultures of E. coli, Klebsiella pneumoniae, and V. cholerae, and a mixed culture of Enterococcus faecium and E. faecilis were grown in saline solution to simulate ballast water and were treated with dosages of ferrate ranging from 0.25 to 5.0 mg/L. A ferrate dose of 5 mg/L resulted in complete disinfection of all organisms tested, and smaller dosages were also very effective. Tailing was consistently observed, and the Hom’s model (1972) appeared to most accurately represent the action of ferrate on these organisms. Salinity and pH did not adversely affect results, and regrowth was not a problem. Ferrate shows good potential as an effective disinfectant in the treatment of ballast water. Water Environ. Res., 80, 561 (2008).


Jost G, Zubkov MV, Yakushev E, Labrenz M, Jurgens K (2008) High abundance and dark CO2 fixation of chemolithoautotrophic prokaryotes in anoxic waters of the Baltic Sea. Limnology and Oceanography 53 :14-22

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We determined the abundance and distribution of chemolithoautotrophic prokaryotes in the redoxcline in two basins (Gotland Deep, Landsort Deep) of the central Baltic Sea by combining dark CO2 fixation measurements with flow cytometric cell sorting. Maximum CO2 fixation rates were recorded in sulfidic waters about 20 m below the chemocline. Flow cytometric analyses of deoxyribonucleic acid (DNA) - stained bacterioplankton revealed the existence of at least five different prokaryotic clusters in water samples collected below the chemocline. Dark CO2 fixation in these clusters was determined by flow cytometric sorting after anoxic incubations with (NaHCO3)-C-14 tracer. Two clusters, representing about 30% of total prokaryotes, were responsible for 65% to 100% of the total dark fixation. Calculated cell-specific CO2 fixation rates in the two basins ranged from 3.5 to 24.7 fg C cell(-1) d(-1) and suggested that these clusters are dominated by chemolithoautotrophic prokaryotes. Mean cell-specific fixation rates reached more than 10 fg C cell(-1) d(-1) in most cases, indicating relatively high growth rates (doubling times 1 - 2 d) of chemolithoautotrophic prokaryotes. Our results provide the first evidence of such high cell-specific CO2 uptake and abundance of chemolithoautotrophic prokaryotes in a pelagic marine environment. However, the identity of the organisms as well as the mechanisms fueling CO2 dark fixation in the anoxic zone remain unknown.


Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L (2008) Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington. Isme Journal 2 :696-706

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Redox sensor green (RSG), a novel fluorescent dye from Invitrogen was employed as a tool for realtime detection of microbes metabolically active in situ, in combination with flow cytometry and cell sorting. Lake Washington sediment, an environment known for high rates of methane oxidation, was used as a model, and methylotrophs were targeted as a functional group. We first tested and optimized the performance of the dye with pure methylotroph cultures. Most cells in actively growing cultures were positive for staining, whereas in starved cultures, few cells fluoresced. However, starved cells could be activated by addition of substrate. High numbers of fluorescing cells were observed in a Lake Washington sediment sample, and activation of subpopulations of cells was demonstrated in response to methane, methanol, methylamine and formaldehyde. The fraction of the population activated by methane was investigated in more detail, by phylogenetic profiling. This approach showed that the major responding species were the Methylomonas species, previously isolated from the site, and Methylobacter species that have not yet been cultivated from Lake Washington. In addition, from the methane-stimulated fraction, uncultivated bacterial sequences were obtained that belonged to unclassified Deltaproteobacteria, unclassified Verrucomicrobiles and unclassified Acidobacteria, suggesting that these microbes may also be involved in methane metabolism. The approach was further tested for its utility in facilitating enrichment for functional types that possess specific metabolic activities but resist cultivation. It was demonstrated that in enrichment cultures inoculated with cells that were sorted after stimulation with methane, Methylobacter sequences could be detected, whereas in enrichment cultures inoculated by randomly sorted cells, Methylomonas species quickly outcompeted all other types.


Karayanni H, Christaki U, Van Wambeke F, Thyssen M, Denis M (2008) Heterotrophic nanoflagellate and ciliate bacterivorous activity and growth in the northeast Atlantic Ocean : a seasonal mesoscale study Aquatic Microbial Ecology 51 :169-181

http://www.int-res.com/abstracts/ame/v51/n2/p169-181/

The grazing effect of heterotrophic nanoflagellates (HNF) and ciliates on bacterial production (BP), as well as their growth rates, was studied in winter, spring and autumn 2001 during the French research project Programme Océan Multidisciplinaire Méso-Echelle (POMME) in the northeast Atlantic Ocean (38 to 45° N, 16 to 22° W). The variability of different parameters studied appears to be largely controlled by the seasonal and latitudinal gradients of primary production rather than the strong eddy activity at the mesoscale level in the area. Heterotrophic microbial abundance, biomass and protistan grazing varied temporally, presenting highest values during the phytoplankton bloom, during the spring period and following the northward propagation of the bloom. HNF biomass integrated over the upper 100 m was highest in spring (270 to 850 mg C m–2). Ciliate integrated biomass was generally ≤160 mg C m–2 except in a Tintinnus sp. bloom in a northern anticyclonic eddy (A1) in spring when it reached 637 mg C m–2. HNF and ciliate growth rates varied from 0.2 to 0.7 d–1 and 0.2 to 1.4 d–1, respectively. The fraction of BP consumed by ciliates was generally <10% except in the anticyclonic eddy A1 in spring during a tintinnid bloom when it reached 37% of BP. In conclusion, our data revealed that HNF can remove a large fraction of bacterial production in the northeast Atlantic Ocean (83 ± 27%, average of all sampling sites and seasons). Ciliates transferred less carbon to higher trophic levels than did HNF ; however, episodic high occurrence of large bacterivorous ciliates, primarily tintinnids, increased the role of theses organisms as C-links in the microbial food web.


Keil DE, Mehlmann T, Butterworth L, Peden-Adams MM (2008) Gestational exposure to perfluorooctane sulfonate suppresses immune function in B6C3F1 mice. Toxicological Sciences 103 :77-85

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Perfluorinated alkyl acids (PFAAs) are used in a multitude of applications and are categorized as high-production volume chemicals produced in quantities exceeding 10,000 lbs/year. As a result, widespread exposure has been documented in adults, children, and infants. It is generally accepted that children are more sensitive to the effects of xenobiotic exposures during fetal and postnatal periods of development ; therefore, considerable efforts are required to investigate the potential impact of a model PFAA, perfluorooctane sulfonate (PFOS) on children’s immunological health. Using the pairing of female C57BL/6N mice with male C3H/HeJ, developmental immunotoxicity was evaluated in B6C3F1 pups following oral maternal exposure to PFOS on gestations days 1-17. Exposure levels included 0.1, 1, and 5 mg/kg/day PFOS. Natural killer (NK) cell activity, SRBC IgM plaque assay, CD4/8 lymphocytic subpopulations, nitrite production in peritoneal macrophages, and body/organ weights were evaluated at 4 and 8 weeks of age in F1 pups. No significant dose-responsive changes in maternal or pup body weights, flow cytometry, or macrophage function were observed, yet hepatomegaly was indicated in F1 male pups at 4 weeks of age. Functional deficits were not evident until 8 weeks of age when NK cell function and IgM production were significantly decreased. When compared with females, male pups were more sensitive to the effects of PFOS thereby establishing a no observed adverse effect level and low observed adverse effect level of 0.1 and 1.0 mg/kg/day (males only) following maternal PFOS exposure level, respectively. This study establishes that the developing immune system is sensitive to the effects of PFOS and results in functional deficits in innate and humoral immunity detectable at adulthood.


Kim EJ, Angell S, Janes J, Watanabe CM (2008) Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection : biotin biosynthesis in the marine microorganism Chromohalobacter. Mol Biosyst 4 :606-613

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18493659

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor ? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.


Larsen A, Tanaka T, Zubkov MV, Thingstad TF (2008) P-affinity measurements of specific osmotroph populations using cell-sorting flow cytometry. Limnology and Oceanography-Methods 6 :355-363

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To elucidate the role that the marine microbes play in global nutrient cycling, it is necessary to recognize how various phyto- and bacterioplankton groups compete for limiting nutrients. Specific phosphate affinity describes an organism’s ability to harvest phosphate at low concentrations from the surrounding water. For the first time, we have taken advantage of cell-sorting flow cytometry in combination with radio-labeled phosphorus to measure this feature of specific osmotrophic groups in natural communities. Specific phosphate affinities for Synechococcus spp. and picoeukaryotes were measured using live, unstained cells. The results were always lower than theoretical calculated maximum values, corresponding well with observations of P-deficiency, or sub-optimal P supply for the osmotroph community, at the time of investigation. Fixing and staining cells before flow sorting offers the advantage of better separation of phytoplankton and showed high sorting reproducibility when applied to nonaxenic Synechococcus cultures. A subsequent investigation of P-leakage from isotopically labeled, fixed, stained cells in nonaxenic cultures of Synechococcus showed that it was only slightly larger than the loss of 17% found when uptake of new label was stopped with adding "cold" phosphate. Possible applications of the currently developed methodology for population specific P affinity measurements by flow sorting are discussed.


Larsen JB, Larsen A, Thyrhaug R, Bratbak G, Sandaa RA (2008) Response of marine viral populations to a nutrient induced phytoplankton bloom at different pCO(2) levels. Biogeosciences 5 :523-533

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During the PeECE III mesocosm experiment in 2005 we investigated how the virioplankton community responded to increased levels of nutrients (N and P) and CO2. We applied a combination of flow cytometry, Pulsed Field Gel Electrophoresis and degenerate PCR primers to categorize and quantify individual viral populations, and to investigate their temporal dynamics. Species specific and degenerate primers enabled us to identify two specific large dsDNA viruses, EhV and CeV, infecting the haptophytes Emiliania huxleyi and Crysochromulina ericina, respectively. Some of the viral populations detected and enumerated by flow cytometry did not respond to altered CO2-levels, but the abundance of EhV and an unidentified dsDNA virus decreased with increasing CO2 levels. Our results thus indicate that CO2 conditions, or the related change in pH, may affect the marine pelagic food web at the viral level. Our results also demonstrate that in order to unravel ecological problems as how CO2 and nutrient levels affect the relationship between marine algal viruses and their hosts, we need to continue the effort to develop molecular markers used to identify both hosts and viruses.


Le Gall F, Rigaut-Jalabert F, Marie D, Garczarek L, Viprey M, Gobet A, Vaulot D (2008) Picoplankton diversity in the South-East Pacific Ocean from cultures. Biogeosciences 5 :203-214

<Go to ISI> ://000253673100005

In late 2004, the BIOSOPE cruise sailed between the equatorial influenced waters off the Marquesas Islands and the nutrient enriched waters of the Chilean upwelling. Along the way, it explored the Southeast Pacific gyre centred around Easter Island, which is probably the most oligotrophic oceanic region on earth. During this cruise, we undertook a vigorous effort to isolate novel photosynthetic picoplanktonic eukaryotes. Two strategies were attempted on board : enrichment of filtered samples with culture medium and sorting of specific populations by flow cytometry based on size and chlorophyll fluorescence. Over 1900 pre-cultures were started and then further purified by flow cytometry, serial dilution or pipette isolation to yield a total of 212 strains. These strains were characterized morphologically and for more than 50% of them, genetically, through partial sequencing of the 18 S rRNA gene.
Among the characterized strains, the largest number belongs to stramenopiles (Heterokontophyta) with a record of 38 strains belonging to the species Pelagomonas calceolata (Pelagophyceae). Strains from the recently described genera Bolidomonas and Florenciella have been re-isolated for the first time since their description. Two other abundant groups are the Chlorophyta, especially Prasinophyceae, and the Haptophyta, especially the genera Phaeocystis and Emiliania. A limited number of heterotrophic flagellates have also been isolated, all of them belonging to groups containing known species. Finally, over a dozen of unicellular cyanobacterial Synechococcus strains have been obtained, some forming unusual short chains.
Overall our strategy was quite successful since it allowed us to isolate a large number of picoplankton strains. Still it failed in two respects. First, apparently very few novel taxa have been obtained. One set of strains is related to Prasinoderma coloniale (Prasinococcales, Prasinophyceae) but their sequences are sufficiently different from the latter to probably belong to a new genus or species. The sequences of two other strains, unfortunately later lost, were phylogenetically affiliated to stramenopile environmental sequences, probably corresponding to a new algal class. Second, very few strains have been obtained from the very oligotrophic central gyre itself. In order to be successful, future work in similar waters should probably combine flow cytometry sorting with culture media and cultivation approaches specifically developed for oligotrophic water species.


Lee BK, Katano T, Kitamura SI, Oh MJ, Han MS (2008) Monitoring of algicidal bacterium, Alteromonas sp strain A14 in its application to natural Cochlodinium polykrikoides blooming seawater using fluorescence in situ hybridization. Journal of Microbiology 46 :274-282

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The red tide of dinoflagellate, Cochlodinium polykrikoides has frequently occurred in coastal waters, causing severe damage to fisheries. In the present study, the algicidal bacterium Alteromonas sp. A14 isolated from the southern coast of Korea was applied to a red tide of C. polykrikoides in a laboratory experiment. In the experiment, the abundance of the strain A14 was monitored using fluorescence in situ hybridization. Inoculation of the A14 at a final cell density of 9.0x10(5) cells/ml caused a significant decrease in C. polykrikoides abundance from 1,830 to 700 cells/ml during 2 days, while abundances of harmless diatoms rapidly increased from 3 days. Abundances of both A14 and other bacteria increased to I day. After I day, with flagellate abundance increased, bacterial abundance decreased. Finally, algicidal bacterial abundance decreased to 3.5x10(4) cells/ml. In the biological control of harmful algal blooms, in addition to decrease in target algal abundance and not occurrence of other harmful blooms, decrease in abundance of utilized organism is also important. This study emphasizes the importance of monitoring the inoculated bacterium when applying bacterium to natural seawater.


Liu H, Zhao M, Zhang C, Ma Y, Liu W (2008) Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253 :89-96

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18822338

Synthetic pyrethroids (SPs) are used in preference to organochlorines and organophosphates due to their high efficiency, low toxicity to mammals, and ready biodegradability. Previous studies reported that enantioselective toxicity of SPs occurs in aquatic toxicity. Several studies have indicated that SPs could lead to oxidative damage in humans or animals which was associated with their toxic effects. Little is known about the differences in the effects of chronic toxicity induced by individual stereoisomers of chiral SPs. The present study was therefore undertaken to evaluate the enantioselectivity in cytotoxicity, genotoxicity caused by bifenthrin (BF) on human amnion epithelial (FL) cell lines and pesticidal activity on target organism. The cell proliferation and cytoflow analysis indicated that 1S-cis-BF presented more toxic effects than 1R-cis-BF above the concentration of 7.5 mg L(-1) (p>0.05). FL cells incubated with 1S-cis-BF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS). In the comet assay, the number of cells with damaged DNA incubated with 1S-cis-BF was more than that with 1R-cis-BF (p<0.01). While the LC(50) values of enantiomer to the target pest on Pieris rapae L. show that 1R-cis-BF was 300 times more active than 1S-cis-BF. These results indicate that the enantioselective toxicity and activity of BF between non-target organism and target organism was reversal. These implications together suggest that assessment of the environmental safety and new pesticides development with chiral centers should consider enantioselectivity.


Llewellyn CA, Tarran GA, Galliene CP, Cummings DG, De Menezes A, Rees AP, Dixon JL, Widdicombe CE, Fileman ES, Wilson WH (2008) Microbial dynamics during the decline of a spring diatom bloom in the Northeast Atlantic. Journal of Plankton Research 30 :261-273

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The microbial dynamics during a spring diatom bloom decline was monitored in the Northeast Atlantic during a 5-day Lagrangian study (8-12 April 2002). Phytoplankton abundance, composition and health status were related to viral and bacterial abundance, zooplankton abundance and grazing rates, as well as bacterial production. Phytoplankton reached maximum concentration on Day 3 (Chl a > 5 mu g L-1) and declined on Day 5 (Chl a similar to 2 mu g L-1) and was dominated (70% of Chl a) by diatoms. Bacterial production increased substantially to > 20 mu g C L-1 day(-1) on Day 3 and concomitantly large viruses decreased in number by half to < 10 x 10(3) mL(-1). This was followed by a 5-fold increase in large viruses on Day 5, indicating infection and subsequent lysis on Days 3 and 5, respectively. Micro- and mesozooplankton grazing were not the principal cause for the decline of the bloom and pheophorbide-a showing little variation in concentration from Days 1-4 (similar to 100 ng L-1) although doubled on Day 5. The poor physiological status of the diatoms, indicated by the high chlorophyllide-a concentrations (50-480 ng L-1), likely promoted a series of closely interrelated events involving bacteria and viruses leading to the demise of the diatom bloom.


Lopes da Silva T, Reis A (2008) The use of multi-parameter flow cytometry to study the impact of n-dodecane additions to marine dinoflagellate microalga Crypthecodinium cohnii batch fermentations and DHA production. J Ind Microbiol Biotechnol 35 :875-887

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18461374

The physiological response of Crypthecodinium cohnii batch cultivations and docosahexaenoic acid (DHA) production to n-dodecane additions were studied. Different n-dodecane concentrations [0, 0.5, 1, 2.5, 5, 10 and 20% (v/v)] were added to preliminary shake flask cultivations. The n-dodecane fraction that gave best results in terms of biomass and DHA production was 0.5% (v/v). The n-dodecane fractions of 2.5, 5, 10 and 20% (v/v) to C. cohnii preliminary shake flask cultures inhibited the microalgal growth and DHA production, although a high proportion of cells with intact cytoplasmic membrane was present in the end of these fermentations. After the addition of a pulse of n-dodecane (0.5% v/v) to C. cohnii exponential growing cells in a bioreactor, glucose uptake volumetric rate increased 2.5-fold, while biomass production volumetric rate increased 2.8-fold. The specific growth rate was increased 1.5-fold. The DHA % in biomass, DHA % of TFA and DHA concentration also increased (54, 22 and 58%, respectively), after the n-dodecane addition. At this n-dodecane fraction (0.5% v/v), multi-parameter flow cytometry demonstrated that C. cohnii cell membrane integrity was not affected. The results demonstrated that the addition of 0.5% of n-dodecane (v/v) to C. cohnii fermentations can be an easy and cheap way for enhancing the biomass and DHA production, avoiding the use of high speed rates (resulting in important power agitation costs) that affects the microalga proliferation and increases the bioprocess costs. A new strategy to improve the DHA production from this microalga in two-phase large-scale bioreactors is now in progress.


Lu AJ, Li ZQ, Zhang QY (2008) Detection of cutaneous antibodies in excised skin explants from grass carp, Ctenopharyngodon idella (Valenciennes), immune to Scophthalmus maximus rhabdovirus. Journal of Fish Diseases 31 :559-565

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This study determined whether cutaneous antibodies were present in excised skin explants of grass carp, Ctenopharyngodon idella, immune to Scophthalmus maximus rhabdovirus (SMRV). Culture fluid from immune skin explants were assayed by indirect enzyme-linked immunosorbent assay (iELISA), Western blot, indirect immunofluorescent assay (IFA) and flow cytometry (FCM). iELISA showed that cutaneous antibody titres were much lower (1:12) than antiserum titres (1:1458) from intraperitoneally immunized grass carp. The phosphoprotein and matrix protein antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using Western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected epithelioma papulosum cyprini (EPC) cells by IFA. FCM showed that 4.39% SMRV-infected EPC cells were detected, while non-specific reaction was seen in 2% of control cells. This is the first description of cutaneous antibodies against SMRV in grass carp.


Ma Y, Zeng Y, Jiao N, Shi Y, Hong N (2008) Vertical distribution and phylogenetic composition of bacteria in the Eastern Tropical North Pacific Ocean. Microbiol Res
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The vertical community structure of bacteria along a depth profile in the Eastern Tropical North Pacific Ocean (13 degrees N, 104 degrees W) was studied by flow cytometry measurement and 16S rRNA gene clone libraries analysis. Picoeukaryotes and Synechococcus peaked at 30m and decreased sharply below 50m, while Prochlorococcus peaked at both 30 and 100m layers and disappeared below 200m. Heterotrophic bacteria peaked above shallow thermocline and decreased along the depth profile. Sequences of total 322 clones from four clone libraries (10, 100, 1000, and 3000m) clustered into nine major lineages. gamma-Proteobacteria dominated all the depths and occupied almost the whole bacterial community at the 3000m. alpha-Proteobacteria was abundant throughout the water column except near the sea bottom, and delta-Proteobacteria peaked at the 1000m depth. Cyanobacteria were primarily limited to the photic zone, and the genetic diversity of Prochlorococcus showed a good correlation with niche adaptation. The appearance of the Cytophaga-Flexibacter-Bacteroides (CFB) group did not show a clear relationship with depth. Actinobacteria were found both in the photic zone and in deep water. Planctomyetes, Acidobacteria, and Verrucomicrobia were present as minor groups and more dominant in the deeper layers of water.


Manti A, Boi P, Falcioni T, Canonico B, Ventura A, Sisti D, Pianetti A, Balsamo M, Papa S (2008) Bacterial cell monitoring in wastewater treatment plants by flow cytometry. Water Environ Res 80 :346-354

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18536486

The activated sludge process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment, in which bacteria constitute the majority and represent the main microorganisms responsible for the degradation process in a plant. In this work, we monitored bacterial charge in different wastewater treatment plants by flow cytometry, also evaluating chlorination effects on bacterial viability, both by flow cytometry and traditional plate counts. Maximum values of bacterial charge were registered in the aeration tank of all plants monitored. Cell viability did not show significant differences (p > 0.05) in samples collected in "before chlorination" and "wastewater effluent" treatment steps ; this suggests that the chlorination was not able to decrease total viable bacterial charge. In this work, we discuss the need to improve microbiological analyses, both in terms of measuring other potential pathogens and of using new methodological approaches in the traditional evaluation of the microbiological quality of effluents.


Martin AP, Zubkov MV, Fasham MJ, Burkill PH, Holland RJ (2008) Microbial spatial variability : An example from the Celtic Sea. Progress in Oceanography 76 :443-465

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In July 2004, dominant populations of microbial ultraplankton (<5 mu m), in the surface of the Celtic Sea (between UK and Eire), were repeatedly mapped using flow cytometry, at 1.5 km resolution over a region of diameter 100 km. The numerically dominant representatives of all basic functional types were enumerated including one group of phototrophic bacteria (Syn), two groups of phytoplankton (PP, NP), three groups of heterotrophic bacterioplankton (HB) and the regionally dominant group of heterotrophic protists (HP).
The distributions of all organisms showed strong spatial variability with little relation to variability in physical fields such as salinity and temperature. Furthermore, there was little agreement between distributions of different organisms. The only linear correlation consistently explaining more than 50% of the variance between any pairing of the organism groups enumerated is between two different groups of HB. Specifically, no linear, or non-linear, relationship is found between any pairings of SYB, PP or HB groups with their protist predators HP. Looking for multiple dependencies, factor analysis reveals three groupings : Syn, PP and low nucleic acid content HB (LNA) ; high nucleic acid content HB (HNA) ; HP and NP. Even the manner in which the spatial variability of Syn, PP and HB abundance varies as a function of length-scale (represented by a semivariogram) differs significantly from that for HP. In summary, although all microbial planktonic groups enumerated are present and numerically dominant throughout the region studied, at face value the relationships between them seem weak.
Nevertheless, the behaviour of a simple, illustrative ecological model, with strongly interacting phototrophs and heterotrophs, with stochastic forcing, is shown to be consistent with the observed poor correlations and differences in how spatial variability varies with lengthscale. Thus, our study suggests that a comparison of microbial abundances alone may not discern strong underlying trophic interactions. Specific knowledge of these processes, in particular grazing, will be required to explain the causes of the observed microbial spatial variability and its resulting consequences for the functioning of the ecosystem. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.


Mary I, Tarran GA, Warwick PE, Terry MJ, Scanlan DJ, Burkill PH, Zubkov MV (2008) Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean. Fems Microbiology Ecology 63 :36-45

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S-35-Methionine and H-3-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.


Masquelier S, Vaulot D (2008) Distribution of micro-organisms along a transect in the South-East Pacific Ocean (BIOSOPE cruise) using epifluorescence microscopy. Biogeosciences 5 :311-321

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The distribution of selected groups of micro-organisms was analyzed along a South-East Pacific Ocean transect sampled during the BIOSOPE cruise in 2004. The transect could be divided into four regions of contrasted trophic status : a High Nutrient Low Chlorophyll (HNLC) region (mesotrophic) near the equator, the South-East Pacific Ocean gyre (hyper-oligotrophic), a transition region between the gyre and the coast of South America (moderately oligotrophic), and the Chile upwelling (eutrophic). The abundance of phycoerythrin containing picocyanobacteria (PE picocyanobacteria), autotrophic and heterotrophic eukaryotes (classified into different size ranges), dinoflagellates, and ciliates was determined by epifluorescence microscopy after DAPI staining. Despite some apparent loss of cells due to sample storage, distribution patterns were broadly similar to those obtained by flow cytometry for PE picocyanobacteria and picoeukaryotes. All populations reached a maximum in the Chile upwelling and a minimum near the centre of the gyre. The maximum abundance of PE picocyanobacteria was 70 10(3) cell mL(-1). Abundance of autotrophic eukaryotes and dinoflagellates reached 24.5 10(3) and 20 cell mL(-1), respectively. We observed a shift in the size distribution of autotrophic eukaryotes from 2-5 mu m in eutrophic and mesotrophic regions to less than 2 mu m in the central region. The contribution of autotrophic eukaryotes to total eukaryotes was the lowest in the central gyre. Maximum concentration of ciliates (18 cell mL(-1)) also occurred in the Chile upwelling, but, in contrast to the other groups, their abundance was very low in the HNLC zone and near the Marquesas Islands. Two key findings of this work that could not have been observed with other techniques are the high percentage of PE picocyanobacteria forming colonies in the HLNC region and the observation of numerous dinoflagellates with bright green autofluorescence.


Mi TY, Yan XJ, Chen HM, Lin J, Wang F, Xu WF (2008) [Proliferation inhibition of lambda-carrageenan oligosaccharides on HUVEC and expression of apoptotic relevant genes]. Yao Xue Xue Bao 43 :474-479

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18717333

To study the anti-proliferation effect of lambda-carrageenan oligosaccharides (lambda-CO) on human umbilical vein endothelial cells (HUVECs) and expression of apoptotic relevant genes, the influence of lambda-CO on HUVECs proliferation was measured by MTT assay ; apoptotic rate, cell cycle distribution and the level of active caspase-3 of HUVECs were analyzed using flow cytometry ; the mRNA level of apoptosis related genes was determined by RT-PCR. At a high concentration of 1 mg x mL(-1), lambda-CO significantly inhibited the endothelial cell proliferation. Annexin-V FITC/PI double stain assay showed that when treated with 0, 0.8, 1 mg x mL(-1) of lambda-CO for 24 h, cell apoptotic rates were (1.67 +/- 1.6)%, (11.48 +/- 2.4)% and (13.81 +/- 2.2)%, respectively, when treated for 48 h, cell apoptotic rates were (2.02 +/- 2.3)%, (13.84 +/- 1.9)% and (38.72 +/- 2.5)%, respectively, cell cycle assay showed the decrease of cells in G0/G1 phase, and increase in S phase. Furthermore, we observed the level of active caspase-3 increased in a dose-dependent manner at 24 th and 48 th. RT-PCR results indicated that mRNA of TNFalpha, p53, caspase-8 and caspase-3 in cells increased after treated with lambda-CO. lambda-CO induce apoptosis of HUVECs in a dose-dependent way and arrests cells at S phase, which mainly due to the up-regulation of apoptotic genes such as TNFalpha, p53, caspase-8, caspase-3 and increase the level of active caspase-3.


Mikula A, Olas M, Sliwinska E, Rybczynski JJ (2008) CRYOPRESERVATION BY ENCAPSULATION OF Gentiana spp. CELL SUSPENSIONS MAINTAINS REGROWTH, EMBRYOGENIC COMPETENCE AND DNA CONTENT. Cryoletters 29 :409-418

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A reliable technique for cryopreservation by encapsulation was developed for two suspension cultures of gentian species (Gentiana tibetica and G. cruciata) of different ages and embryogenic potential. The,effect of water content, aggregate size and the subculture time on viability was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) test. Regrowth of a proembryogenic mass (PEM) on agar, liquid or agar/liquid media was assayed by measuring the increase in biomass. A water content of 24-30% (fresh weight basis) after 5-6 h dehydration of encapsulated cells of gentians yielded the highest survival (68% for G. tibetica and 83% for G. cruciata) after cryopreservation. Regardless of species, aggregate size and subculture time, the lowest PEM survival was 44%. These parameters did not influence the survival of G. tibetica PEM, but the survival of G. cruciata was higher when the smaller aggregates were cryopreserved on the 5(th) day of culture. Agar/liquid culture caused the greatest biomass increase. Cryopreservation did not affect the characteristics of suspension cultures and their regrowth after thawing, nor the number and dynamics of somatic embryos formed. Flow cytometry showed that cryopreservation did not change the genome size of the PEMs or regenerants.


Mills MM, Moore CM, Langlois R, Milne A, Achterberg E, Nachtigall K, Lochte K, Geider RJ, La Roche J (2008) Nitrogen and phosphorus co-limitation of bacterial productivity and growth in the oligotrophic subtropical North Atlantic. Limnology and Oceanography 53 :824-834

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Bacterial productivity and biomass are thought to be limited by dissolved organic carbon (DOC) in much of the world’s oceans. However, the mixed layer of oligotrophic oceans is often depleted in dissolved inorganic nitrogen and phosphate, raising the possibility that macronutrients may also limit heterotrophic bacterial growth. We used nutrient bioassay experiments to determine whether inorganic nutrients (N, P, Fe) and/or DOC could limit bacterial productivity and biomass in the central North Atlantic during the spring of 2004 (Mar-Apr). We observed that both heterotrophic bacterial productivity and biomass were co-limited by N and P in the oligotrophic North Atlantic, and additions of labile DOC (glucose) provided no stimulation unless N and P were also added. Flow cytometry results indicated that only a small subset of large cells high in nucleic acid content were responsible for the increased productivity in the combined NP amendments. In contrast, nutrient additions elicited no net change on the dominant component of the bacterial population, composed of small cells with relatively low nucleic acid content. In the combined NP treatments the relative increase in bacterial production was greater than that measured when phytoplankton productivity was relieved of nitrogen limitation. These results suggest that N and P co-limitation in the bacterial community results in increased competition between the heterotrophic and autotrophic components of the surface communities in the Central North Atlantic Ocean, and potentially impacts the cycling of organic matter by the bacterioplankton.


Minnhagen S, Carvalho WF, Salomon PS, Janson S (2008) Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty. Environmental Microbiology 10 :2411-2417

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Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 +/- 340 copies of nuclear rDNA and 1300 +/- 200 copies of plastid rDNA ; cells from the G2 group contained 9700 +/- 58 copies of nuclear rDNA and 1400 +/- 220 copies of plastid rDNA (mean +/- SD, n = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.


Nishimura M, Shimakita T, Matsuzaki T, Tashiro Y, Kogure K (2008) Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus. Fisheries Science 74 :405-410

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The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast (Candida albicans) and a bacterium (Escherichia coli) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.


Not F, Latasa M, Scharek R, Viprey M, Karleskind P, Balague V, Ontoria-Oviedo I, Cumino A, Goetze E, Vaulot D, Massana R (2008) Protistan assemblages across the Indian Ocean, with a specific emphasis on the picoeukaryotes. Deep-Sea Research Part I-Oceanographic Research Papers 55 :1456-1473

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Protists, and among them the picoeukaryotes (cells <3 mu m), have been described as significant contributors to both carbon biomass and production in oligotrophic regions of the oceans. However, protist assemblages remain largely undescribed in pelagic ecosystems and in particular in the Indian Ocean. In the present work, we investigated protists along an eastward transect across the sub-tropical gyre of the Indian Ocean (from South Africa to Australia), with a particular focus on picoeukaryotes. We combined inverted and epifluorescence microscopy, flow cytometry, pigment analysis, denaturing get gradient electrophoresis (DGGE), 18S rDNA clone libraries, and fluorescent in situ hybridization (FISH). Overall the picophytoplankton fraction contributed 88% and 90% of total Chl a at the surface and DCM, respectively, with picoeukaryotes accounting for 38% and 50% of total Chl a at the surface and DCM. Considering only the Indian South Subtropical Gyre (ISSG) province, we observed greater shifts in the picoeukaryotic assemblage throughout the upper 200 in of the water column than along the ca. 10,000 kin cruise track. In terms of taxonomic diversity and contribution of each taxon to the picoeukaryotic community, prasinophytes were well represented at more coastal stations with the genus Micromonas reaching densities up to 750 cell mL(-1) in coastal waters and less than 100 cell mL(-1) at open ocean stations. Haptophytes (56% and 45% of picoeukaryotic pigments at surface and DCM, respectively) and possibly pelagophytes (28% and 40% of picoeukaryotic pigments at surface and DCM, respectively) appeared to be dominant at open ocean stations. Other groups and in particular organisms affiliated to chrysophytes, and to a lesser extent to cryptophytes, appear as clear targets for future qualitative and quantitative studies. Moreover, the occurrence of many sequences related to radiolarians (5% and 27% at surface and DCM, respectively) will require further investigation. (C) 2008 Elsevier Ltd. All rights reserved.


Parada V, Baudoux AC, Sintes E, Weinbauer MG, Herndl GJ (2008) Dynamics and diversity of newly produced virioplankton in the North Sea. Isme Journal 2 :924-936

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Viral diversity has been studied in a variety of marine habitats and spatial and seasonal changes have been documented. Most of the bacteriophages are considered host specific and are thought to affect fast growing prokaryotic phylotypes more than slow growing ones. We hypothesized that viral infection and consequently, lysis occurs in pulses with only a few prokaryotic phylotypes lysed at any given time. Thus, we propose that the newly produced viruses represent only a fraction of the viral diversity present at any given time. Virioplankton diversity was assessed by pulsed-field gel electrophoresis in the surface waters of three distinct areas of the North Sea during the spring and summer. Bulk virioplankton diversity was fairly stable in these waters. Viral diversity produced by the indigenous bacterioplankton, however, exhibited day-to-day variability with only a few bands produced at any given time. These bands frequently matched bands of the in situ virioplankton ; however, bands not present in the band pattern of the in situ virioplankton community were also found. These new bands probably indicate infection and subsequent release of viruses from bacterioplankton phylotypes previously not infected by these specific viruses. Overall, our results demonstrate that viral infection and lysis are rather dynamic processes. The main targets of viral infection are changing apparently on time scales of hours to days indicating that viral infection might effectively regulate and maintain bacterioplankton diversity.


Parisi MG, Li H, Jouvet LBP, Dyrynda EA, Parrinello N, Cammarata M, Roch P (2008) Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria. Fish & Shellfish Immunology 25 :834-840

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Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12 h post-injection for Micrococcus lysodeikticus, 24 h for Vibrio splendidus and more than 48 h for V ibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9 h post-injection of living bacteria and -56% 3 h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9 h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12 h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides. (C) 2008 Elsevier Ltd. All rights reserved.


Paterson HL, Knott B, Koslow AJ, Waite AM (2008) The grazing impact of microzooplankton off south west Western Australia : as measured by the dilution technique. Journal of Plankton Research 30 :379-392

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Grazing rates of microzooplankton feeding on picophytoplankton (Flow-cytometry) and total phytoplankton (Chlorophyll a) were measured in the eastern Indian Ocean off south west Western Australia from February 2003 to December 2004. Three sites representing different oceanographic habitats, the coastal lagoon, the outer shelf and the continental slope (1000 m) were sampled. The dilution method of Landry and Hassett (1982, Estimating the grazing impact of marine micro-zooplankton. Mar. Biol., 67, 283-288) was used and analysed by chlorophyll a analysis and flow-cytometry. During summer, the apparent growth rate of total phytoplankton exceeded loss due to microzooplankton grazing in the lagoon and at the outer shelf. On the slope, the phytoplankton assemblage was always dominated by small cells (< 5 mu m). Although their apparent growth rates were also higher in summer, these were matched by increasing microzooplankton grazing rates. Saturated feeding responses at the outer shelf and slope stations during summer were detected. In this low prey, low productivity environment, this response is either a new type of threshold feeding or an artefact of the dilution method which would result in an over-estimate of both phytoplankton growth and microzooplankton grazing.


Payet JP, Suttle CA (2008) Physical and biological correlates of virus dynamics in the southern Beaufort Sea and Amundsen Gulf. Journal of Marine Systems 74 :933-945

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As part of the Canadian Arctic Shelf Exchange Study (CASES), we investigated the spatial and seasonal distributions of viruses in relation to biotic (bacteria, chlorophyll-a (chl a)) and abiotic variables (temperature, salinity and depth). Sampling occurred in the southern Beaufort Sea Shelf in the region of the Amundsen Gulf and Mackenzie Shelf, between November 2003 and August 2004. Bacterial and viral abundances estimated by epifluorescence microscopy (EFM) and flow cytometry (FC) were highly correlated (r(2)=0.89 and r(2)=0.87, respectively), although estimates by EFM were slightly higher (FC=1.08 x EFM+0.12 and FC=1.07 x EFM+0.43, respectively). Viral abundances ranged from 0.13 x 10(6) to 23 x 10(6) ml(-1), and in surface waters were similar to 2-fold higher during the spring bloom in May and June and similar to 1.5-fold higher during July and August, relative to winter abundances. These increases were coincident with a similar to 6-fold increase in chl a during spring and a similar to 4-fold increase in bacteria during summer. Surface viral abundances near the Mackenzie River were similar to 2-fold higher than in the Mackenzie Shelf and Amundsen Gulf regions during the peak summer discharge, concomitant with a similar to 5.5-fold increase in chl a (tip to 2.4 mu g l(-1)) and a similar to 2-fold increase in bacterial abundance (up to 22 x 10(5) ml(-1)). Using FC, two subgroups of viruses and heterotrophic bacteria were defined. A low SYBR-green fluorescence virus subgroup (V2) representing similar to 71% of the total viral abundance, was linked to the abundance of high nucleic acid fluorescence (HNA) bacteria (a proxy for bacterial activity), which represented 42 to 72% of the bacteria in surface layers. A high SYBR-green fluorescence viral subgroup (V1) was more related to high chl a concentrations that occurred in surface waters during spring and at stations near the Mackenzie River plume during the summer discharge. These results suggest that V1 infect phytoplankton, while most V2 are bacteriophages. On the Beaufort Sea shelf, viral abundance displayed seasonal and spatial variations in conjunction with chl a concentration, bacterial abundance and composition, temperature, salinity and depth. The highly dynamic nature of viral abundance and its correlation with increases in chl a concentration and bacterial abundance implies that viruses are important agents of microbial mortality in Arctic shelf waters. (C) 2007 Elsevier B.V. All rights reserved.


Pellisso SC, Munoz MJ, Carballo M, Sanchez-Vizcaino JM (2008) Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro. Veterinary Immunology and Immunopathology 121 :189-198

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Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10 mg/L), Al (2,5, 25 and 50 mg/L), Cd (1, 10, 20 and 40 mg/L), Pb (1, 10, 20 and 50 mg/L) and Cr (I and 10 mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lympboproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to I mg/L of mercury, 10 mg/L of cadmium and 50 mg/L of lead. Decreased phagocytosis was also observed at 5 mg/L of mercury, 50 mg/L of aluminium and 10 mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals. (c) 2007 Elsevier B.V. All rights reserved.


Prince EK, Myers TL, Kubanek J (2008) Effects of harmful algal blooms on competitors : Allelopathic mechanisms of the red tide dinoflagellate Karenia brevis. Limnology and Oceanography 53 :531-541

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Because competitive interactions may have led to adaptations enabling bloom-forming phytoplankton to dominate pelagic communities, we explored the allelopathic effects of one red tide dinoflagellate, Karenia brevis, on competing phytoplankton species. Exposure to waterborne compounds from natural K. brevis blooms resulted in growth inhibition or death for four of five co-occurring species tested, whereas compounds exuded by K. brevis cultures suppressed three of these same competitors (the diatoms Asterionellopsis glacialis and Skeletonema costatum and the dinoflagellate Prorocentrum minimum) plus one additional species (the dinoflagellate Akashiwo cf. sanguinea) that was unaffected by bloom exudates. K. brevis exudates lowered photosynthetic efficiency and damaged cell membranes of competing phytoplankton, but had no effect on competitor esterase activity, nor did they limit competitor access to iron. Overall, during blooms, K. brevis exudes potent allelopathic compounds, competitors vary in their susceptibility to K. brevis allelopathy, and K. brevis may achieve nearly monospecific blooms by lowering the photosynthetic efficiency of competitor species and increasing competitor membrane permeability, eventually resulting in competitor growth suppression or death.


Ribalet F, Intertaglia L, Lebaron P, Casotti R (2008) Differential effect of three polyunsaturated aldehydes on marine bacterial isolates. Aquatic Toxicology 86 :249-255

<Go to ISI> ://WOS:000253696200014

Bioactive polyunsaturated aldehydes (PUAs) are produced by several marine phytoplankton (mainly diatoms) and have been shown to have a detrimental effect on a wide variety of organisms, including phytoplankton and invertebrates. However, their potential impact on marine bacteria has been largely neglected. We assess here the effect of three PUAs produced by marine diatoms : 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal, on the growth of 33 marine bacterial strains, including 16 strains isolated during a bloom of the PUA-producing diatom Skeletonema marinoi in the Northern Adriatic Sea. A concentration-dependent growth reduction was observed for 19 bacterial strains at concentrations ranging from 3 to 145 mu mol L-1. Surprisingly, Eudora adriatica strain MOLA358 (Flavobacteriaceae) and Alteromonas hispanica strain MOLA151 (Alteromonadaceae) showed growth stimulation upon exposure to PUAs at concentrations between 13 and 18 mu mol L-1. The remaining 12 strains were unaffected by even very high PUA concentrations. Strains isolated during the diatom bloom showed remarkable resistance to PUA exposures, with only two out of 16 strains showing growth inhibition at PUA concentrations below 106, 130, and 145 mu mol L-1 for 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal, respectively. No correlation between taxonomical position and sensitivity to PUA was observed. Considering that many bacteria thrive in close vicinity of diatom cells, it is likely that these compounds may shape the structure of associated bacterial communities by representing a selection force. This is even more relevant during the final stages of blooms, when senescence and nutrient limitation increase the potential production and release of aldehydes. (c) 2007 Elsevier B.V. All rights reserved.


Rusiecka I, Skladanowski AC (2008) Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids. Acta Biochimica Polonica 55 :329-337

<Go to ISI> ://000259968500012

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C-6 up to C-9 and decreased for C-10. Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.


Sarmento H, Unrein F, Isumbisho M, Stenuite S, Gasol JM, Descy JP (2008) Abundance and distribution of picoplankton in tropical, oligotrophic Lake Kivu, eastern Africa. Freshwater Biology 53 :756-771

<Go to ISI> ://000253886900009

1. We used flow cytometry to characterize freshwater photosynthetic picoplankton (PPP) and heterotrophic bacteria (HB) in Lake Kivu, one of the East-African great lakes. Throughout three cruises run in different seasons, covering the four major basins, phycoerythrin-rich cells dominated the PPP. Heterotrophic bacteria and PPP cell numbers were always high and spatial variations were modest. This represents an important difference from temperate and high latitude lakes that show high fluctuations in cell abundance over an annual cycle.
2. Three populations of picocyanobacteria were identified : one corresponded to single-cells (identified as Synechococcus by epifluorescence microscopy, molecular methods and pigment content), and the two other that most probably correspond to two and four celled colonies of the same taxon. The proportion of these two subpopulations was greater under stratified conditions, with stronger nutrient limitation.
3. High PPP concentrations (c. 10(5) cell mL(-1)) relative to HB (c. 10(6) cell mL(-1)) were always found. Lake Kivu supports relatively less bacteria than phytoplankton biomass than temperate systems, probably as a consequence of factors such as temperature, oligotrophy, nutrient limitation and trophic structure.
4. A review of PPP concentration across aquatic systems suggests that the abundance of Synechococcus-like cyanobacteria in large, oligotrophic, tropical lakes is very high.
5. Photosynthetic picoplankton cell abundances in the oligotrophic tropical lakes Kivu and Tanganyika are comparable to those of eutrophic temperate lakes. This apparently contradicts the view that PPP abundance increases with increasing eutrophy. More data on PPP in tropical lakes are needed to explore further this particular pattern.


Sawstrom C, Pearce I, Davidson AT, Rosen P, Laybourn-Parry J (2008) Influence of environmental conditions, bacterial activity and viability on the viral component in 10 Antarctic lakes. Fems Microbiology Ecology 63 :12-22

<Go to ISI> ://000251504000003

The influence of biotic and environmental variables on the abundance of virus-like particles (VLP) and lysogeny was investigated by examining 10 Antarctic lakes in the Vestfold Hills, Antarctica, in the Austral Spring. Abundances of viruses and bacteria and bacterial metabolic activity were estimated using SYBR Gold (Molecular Probes), Baclight((TM)) (Molecular Probes) and 6-carboxy fluorescein diacetate (6CFDA). Total bacterial abundances among the lakes ranged between 0.12 and 0.47 x 10(9) cells L-1. The proportion of intact bacteria (SYTO (R) 9-stained cells) ranged from 13.5% to 83.5% of the total while active (6CFDA-stained) bacteria ranged from 33% to 116%. Lysogeny, as determined with Mitomycin C, was only detected in one of the lakes surveyed, indicating that viral replication was occurring predominately via the lytic cycle. Principal component analysis and confirmatory correlation analysis of individual variables showed that high abundances of VLP occurred in lakes of high conductivity with high concentrations of soluble reactive phosphorus and dissolved organic carbon. These lakes supported high concentrations of chlorophyll a, intact bacteria, rates of bacterial production and virus to bacteria ratios. Thus, it was suggested that viral abundance in the Antarctic lakes was determined by the trophic status of the lake and the resultant abundance of intact bacterial hosts.


Somorjai IML, Camasses A, Riviere B, Escriva H (2008) Development of a semi-closed aquaculture system for monitoring of individual amphioxus (Branchiostoma lanceolatum), with high survivorship. Aquaculture 281 :145-150

<Go to ISI> ://000259481400024

The European amphioxus Branchiostoma lanceolatum is becoming an important model for developmental studies, and as such requires more study both in the field and in the laboratory. We present an experimental set-up with temperature and flow rate control that allows easy care and monitoring of individual amphioxus. Over the course of several months, 98/100 individuals in two size categories and experiencing different levels of handling stress survived. Flow cytometry and gut contents indicate that the system meets the nutritional needs of the amphioxus. This simple and effective system for separate aquaculture of individual amphioxus prevents infections due to crowding. It should be particularly useful for future breeding, genetic, behavioural and life history studies, and can be easily adapted to other marine organisms. (C) 2008 Elsevier B.V. All rights reserved.


Souid-Mensi G, Moukha S, Maaroufi K, Creppy EE (2008) Combined cytotoxicity and genotoxicity of a marine toxin and seafood contaminant metal ions (chromium and cadmium). Environ Toxicol 23 :1-8

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18214935

Algal bloom with consequent production of marine toxins contaminating bivalves is increasing in costal regions worldwide because of sea water quality worsening. Contamination of seafood by diarrheic shellfish poisoning toxins (DSP) together with metals is frequently reported, a phenomenon not fully explained yet. In this context, metal ions were assayed in clams collected from the banned area of Boughrara, Tunisia, contaminated by Gymnodinium and other algae such as Dinophysis sp, accumulated by these bivalves. The presence of toxic metals ions such as Chromium (Cr) and Cadmium (Cd) in meat, shells, and water released by the clams prompted us to experiment in Caco-2 intestinal cell line toxic effects of these heavy metals ions in combination with okadaic acid, one DSP present in clams to assess the potential global toxicity. Cr and Cd produce additive effects in (i) reactive oxygen species production, (ii) cytotoxicity as assessed by the mitochondrial activity testing method (MTT test), and (iii) DNA lesions evaluated by agarose gel electrophoresis and acridine orange staining. Exaggerated DNA fragmentation is observed, suggesting an overloading of repair capacity of Caco-2 cells. The apoptosis suggested by a DNA fragment sizing (180-200 bp) in agarose gel and mechanisms underlying these additive effects in Caco-2 cells still need to be more comprehensively explained.


Stachowski-Haberkorn S, Becker B, Marie D, Haberkorn H, Coroller L, de la Broise D (2008) Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment. Aquat Toxicol 89 :232-241

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18760491

The effects of the herbicide Roundup (glyphosate) on natural marine microbial communities were assessed in a 7-day field experiment using microcosms. Bottles were maintained underwater at 6m depth, and 10% of their water content was changed every other day. The comparison of control microcosms and surrounding surface water showed that the microcosm system tested here can be considered as representative of the natural surrounding environment. A temporal temperature gradient gel electrophoresis (TTGE) was run on 16S and 18S rDNA-amplified extracts from the whole microbial community. Cluster analysis of the 16S gel showed differences between control and treatment fingerprints for Roundup at 1 microg L(-1) (ANOSIM, p=0.055 ; R=0.53), and 10 microg L(-1) (ANOSIM, p=0.086 ; R=0.40). Flow cytometry analysis revealed a significant increase in the prasinophyte-like population when Roundup concentration was increased to 10 microg L(-1). This study demonstrates that a disturbance was caused to the marine microbial community exposed to 1 microg L(-1) Roundup concentration, a value typical of those reported in coastal waters during a run-off event.


Stauber JL, Binet MT, Bao VWW, Boge J, Zhang AQ, Leung KMY, Adams MS (2008) Comparison of the QwikLite (TM) algal bioluminescence test with marine algal growth rate inhibition bioassays. Environmental Toxicology 23 :617-625

<Go to ISI> ://000259284000009

Although marine algal bioassays based on growth rate inhibition over 72-96 h have been widely used to assess the toxicity of contaminants in waters and sediments, changes in pH over the test duration can lead to changes in contaminant speciation and consequently an under- or over-estimation of toxicity. In addition, high cell densities are used in order to obtain a detectable response, further reducing the tests’ environmental relevance in marine waters. There is a need for rapid acute tests with ecologically relevant test endpoints that may be used as surrogates for longer-term chronic tests. This study compares the sensitivity and reproducibility of a rapid marine dinoflagellate (Pyrocystis lunula) bioluminescence test (QwikLite (TM)) with standard algal growth rate bioassays (Nitzschia closterium and Entomoneis c.f. punctulata) using ammonia and several antifouling agents (tributyltin [TBT], copper, and diuron) as reference toxicants. QwikLite was of similar sensitivity to ammonia as standard algal growth rate tests, but was less sensitive to copper, diuron and TBT, with 24-h EC50 values of 10 +/- 1.1 mg N/L, 0.128 +/- 0.021 mg Cu/L, 19 +/- 13 mg diuron/L, and 0.226 +/- 0.028 mg TBT/L. Inter-test precision using different batches of P lunula was generally acceptable. On the basis of NOEC values, QwikLite (TM) was more sensitive to copper and ammonia at 25 degrees C than at 21 degrees C. QwikLite (TM) shows promise as a rapid, inexpensive screening test for acute toxicity of contaminants in marine environments. (C) 2008 Wiley Periodicals, Inc.


Takahashi CK, Lourenco NGGS, Lopes TF, Rall VLM, Lopes CAM (2008) Ballast water : A review of the impact on the world public health. Journal of Venomous Animals and Toxins Including Tropical Diseases 14 :393-408

<Go to ISI> ://000259279500002

Since the nineteenth century ships have been using ballast water (BW) for safety, stability, propulsion and maneuverability, as well as to redress loss of fuel weight and water consumption, and to maintain structural stress at acceptable levels. Ballast water has been spreading many non-native species around the globe, but little is known about the extent and potential significance of ship-mediated transfer of microorganisms. The global movements of ballast water by ships create a long-distance dispersal mechanism for human pathogens that may be important in the worldwide distribution of microorganisms, as well as for the epidemiology of waterborne diseases. Only a few studies have been carried out on this subject, most of them involving ballast water containing crustacean larvae and phytoplankton. Specialized microbiological studies on these waters are necessary to avoid a repeat of what happened in 1991, when epidemic cholera was reported in Peru and rapidly spread through Latin America and Mexico. In July of 1992, Vibrio cholerae was found in the USA and the Food and Drug Administration (FDA) determined that it came from ballast water of ships whose last port of call was in South America. In Brazil, just a few studies about the subject have been performed. An exploratory study by the Brazilian National Health Surveillance Agency (Agencia Nacional de Vigilancia Sanitaria - ANVISA) found in ballast water different microorganisms, such as fecal coliforms, Escherichia coli, Enterococcus faecalis, Clostridium perfringens, coliphages, Vibrio cholerae O1 and Vibrio cholerae non-O1. Until now, Brazil has been focusing only on organisms transported to its territory from other countries by ballast water, to avoid their establishment and dissemination in Brazilian areas. Studies that can assess the probability that water ballast carries pathogenic microorganisms are extremely important, as is the examination of ships that arrive in the country. Treatment of the human infections caused by BW exists but none is completely safe and efficient.


Teske A, Sorensen KB (2008) Uncultured archaea in deep marine subsurface sediments : have we caught them all ? Isme Journal 2 :3-18

<Go to ISI> ://000252592100002

Deep marine subsurface sediments represent a novel archaeal biosphere with unknown physiology ; the sedimentary subsurface harbors numerous novel phylogenetic lineages of archaea that are at present uncultured. Archaeal 16S rRNA analyses of deep subsurface sediments demonstrate their global occurrence and wide habitat range, including deep subsurface sediments, methane seeps and organic-rich coastal sediments. These subsurface archaeal lineages were discovered by PCR of extracted environmental DNA ; their detection ultimately depends on the specificity of the archaeal PCR 16S rRNA primers. Surprisingly high mismatch frequencies for some archaeal PCR primers result in amplification bias against the corresponding archaeal lineages ; this review presents some examples. Obviously, most archaeal 16S rRNA PCR primers were developed either before the discovery of these deep subsurface archaeal lineages, or without taking their sequence variants into account. PCR surveys with multiple primer combinations, revision and updates of primers whenever possible, and increasing use of PCR-independent methods in molecular microbial ecology will contribute to a more comprehensive view of subsurface archaeal communities.


Thyssen M, Garcia N, Denis M (2008) Sub meso scale phytoplankton distribution in the north east Atlantic surface waters determined with an automated flow cytometer. Biogeosciences Discussion 5 :2471–2503

http://biogeosciences-discuss.net/5/2471/2008/bgd-5-2471-2008.pdf

Phytoplankton cells in the size range -1–50 µm were analysed in surface waters using
an automated flow cytometer, the Cytosub (http://www.cytobuoy.com), from the Azores
to the French Brittany during spring 2007. The Cytosub records the pulse shape of the
optical signals generated by phytoplankton cells when intercepted by the laser beam. 5
A total of 6 distinct optical groups were resolved during the whole transect, and the high
frequency sampling (15 min) provided evidence for the cellular cycle (based on cyclic
changes in cell size and fluorescence) and distribution changes linked to the different
water characteristics crossed in the north east Atlantic provinces. Nutrient concentrations
and mixed layer depth varied from west to east, with a decrease in the mixed layer 10
depth and high nutrient concentrations in the middle of the transect as well as near the
French coast. Data provided a link between the sub meso scale processes and phytoplankton
patchiness, some abundance variations due to the cellular cycle can be
pointed out. The high frequency spatial sampling encompasses temporal variations of
the phytoplankton abundance, offering a better insight into phytoplankton distribution.


Thyssen M, Mathieu D, Garcia N, Denis M (2008) Short-term variation of phytoplankton assemblages in Mediterranean coastal waters recorded with an automated submerged flow cytometer. Journal of Plankton Research 30 :1027-1040

<Go to ISI> ://000259304000006

Short-term variations of phytoplankton communities are poorly documented. To overcome these limitations and make observations on a short-time (hours) scale, we moored a submersible flow cytometer (CytoBuoy b.v.) in the Bay of Marseille. The CytoSub monitored phytoplankton every 30 min at a fixed site (2 m depth) during summer 2005. The data treatment, conducted on the basis of pulse-shape analysis, resolved seven clusters. Daily sampling of nutrients and continuous information on salinity, temperature and wind speed allowed distinction between diel cycles and the impact of environmental factors on phytoplankton communities. Autocorrelation of the time series showed a significant periodicity of similar to 24 h for most of the clusters during undisturbed meteorological conditions. Two clusters had regular daily abundance variations in the range 0->10(3) cells cm(-3). Two strong wind events revealed similar cluster succession patterns occurring over several days after the wind events. These results provided by the high frequency in situ analysis suggest that the flow cytometry resolved clusters, showing independent behaviour and distinct environment-correlated variations, which may be considered as functional groups. We point out its potential for global oceanic observing systems for which such systems could provide real biological information.


Thyssen M, Tarran GA, Zubkov MV, Holland RJ, Gregori G, Burkill PH, Denis M (2008) The emergence of automated high-frequency flow cytometry : revealing temporal and spatial phytoplankton variability. J Plankton Res 30 :333-343

http://plankt.oxfordjournals.org/cgi/content/abstract/30/3/333

Phytoplankton observation is the product of a number of trade-offs related to sampling processes, required level of diversity and size spectrum analysis capabilities of the techniques involved. Instruments combining the morphological and high-frequency analysis for phytoplankton cells are now available. This paper presents an application of the automated high-resolution flow cytometer Cytosub as a tool for analysing phytoplanktonic cells in their natural environment. High resolution data from a temporal study in the Bay of Marseille (analysis every 30 min over 1 month) and a spatial study in the Southern Indian Ocean (analysis every 5 min at 10 knots over 5 days) are presented to illustrate the capabilities and limitations of the instrument. Automated high-frequency flow cytometry revealed the spatial and temporal variability of phytoplankton in the size range 1-[ ]50 microm that could not be resolved otherwise. Due to some limitations (instrumental memory, volume analysed per sample), recorded counts could be statistically too low. By combining high-frequency consecutive samples, it is possible to decrease the counting error, following Poisson’s law, and to retain the main features of phytoplankton variability. With this technique, the analysis of phytoplankton variability combines adequate sampling frequency and effective monitoring of community changes.


Tijdens M, Hoogveld HL, Kamst-van Agterveld MP, Simis SG, Baudoux AC, Laanbroek HJ, Gons HJ (2008) Population dynamics and diversity of viruses, bacteria and phytoplankton in a shallow eutrophic lake. Microb Ecol 56 :29-42

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17924158

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 x 10(7) and 1.3 x 10(8) virus-like particles ml(-1) and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus-host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht.


Tijdens M, Hoogveld HL, Kamst-van Agterveld MP, Simis SGH, Baudoux AC, Laanbroek HJ, Gons HJ (2008) Population dynamics and diversity of viruses, bacteria and phytoplankton in a shallow eutrophic lake. Microbial Ecology 56 :29-42

<Go to ISI> ://000256472700004

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 x 10(7) and 1.3 x 10(8) virus-like particles ml(-1) and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus-host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht.


Tijdens M, van de Waal DB, Slovackova H, Hoogveld HL, Gons HJ (2008) Estimates of bacterial and phytoplankton mortality caused by viral lysis and microzooplankton grazing in a shallow eutrophic lake. Freshwater Biology 53 :1126-1141

<Go to ISI> ://000255713100006

1. Since viral lysis and zooplankton grazing differ in their impact on the aquatic food web, it is important to assess the relative importance of both mortality factors. In this study, an adapted version of the dilution technique was applied to simultaneously estimate the impact of both viral lysis and zooplankton grazing on the mortality of heterotrophic bacteria, eukaryotic algae, unicellular cyanobacteria, prochlorophytes and especially filamentous cyanobacteria in a shallow eutrophic lake.
2. Four dilution experiments were performed in December 2004, January 2005, and March and April 2006. Viral and heterotrophic bacterial abundances were obtained by epifluorescence microscopy and abundances of different phytoplankton groups by flow cytometry and light microscopy.
3. Viral lysis was identified as the main mortality cause during the December 2004 and January 2005 experiments, apparently removing between 84% and 97% of the potential filamentous cyanobacterial production and up to 101% of the potential heterotrophic bacterial production. Microzooplankton grazing was estimated to remove between 90% and 99% of the potential unicellular cyanobacterial production and up to 46% of the potential heterotrophic bacterial production during the spring 2006 experiments.
4. In some cases, no significant impact of viral lysis or zooplankton grazing was detected. Contrary to expectations, the apparent growth rate of filamentous cyanobacteria was even sometimes observed to decrease significantly upon dilution of microzooplankton.
5. The dilution technique can give valuable insight into the impact of zooplankton grazing and viral lysis on the mortality of different plankton groups but require some caveats require special care to be taken when comparing and interpreting results.


Travers MA, Barbou A, Le Goic N, Huchette S, Paillard C, Koken M (2008) Construction of a stable GFP-tagged Vibrio harveyi strain for bacterial dynamics analysis of abalone infection. FEMS Microbiol Lett 289 :34-40

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19054091

Vibrio harveyi is a bacterial marine pathogen that can cause fatal disease in a large range of vertebrates and invertebrates, including the commercially important marine gastropod, Haliotis tuberculata. Since 1997, strains of this bacterium have regularly been causing high mortalities in farmed and wild abalone populations. The way in which the pathogen enters into abalone and the disease transmission mechanisms are thus far unknown. Therefore, a pathogenic strain, ORM4, was green fluorescent protein-tagged and validated both for its growth characteristics and for its virulence as a genuine model for abalone disease. The strain allows V. harveyi quantification by flow cytometry in seawater and in abalone haemolymph as well as the in situ detection of the parasite inside abalone tissues.


Vazquez-Dominguez E, Duarte CM, Agusti S, Jurgens K, Vaque D, Gasol JM (2008) Microbial plankton abundance and heterotrophic activity across the Central Atlantic Ocean. Progress in Oceanography 79 :83-94

<Go to ISI> ://000261023400005

The role of microorganisms in the transfer of carbon of marine systems is very important in open oligotrophic oceans. Here, we analyze the picoplankton structure, the heterotrophic bacterioplankton activity, and the predator-prey relationships between heterotrophic bacteria and nanoflagellates during two large scale cruises in the Central Atlantic Ocean (similar to 29 degrees N to similar to 40 degrees S). Latitud cruises were performed in 1995 between March-April and October-November. During both cruises we crossed the regions of different trophic statuses ; where we measured different biological variables both at the surface and at the deep chlorophyll maximum (DCM). The concentration of chlorophyll a varied between 0.1 and 0.8 mg m(-3), the abundance of heterotrophic bacteria varied between <1.0 x 10(5) and >1.0 x 10(6) cells ml(-1), and that of heterotrophic nanoflagellates between <100 and >1.0 x 10(4) cells ml(-1). The production of heterotrophic bacteria varied more than three orders of magnitude between <0.01 and 24 mu gC L-1 d(-1) ; and the growth rates were in the range <0.01-2.1 d(-1). In the Latitud-II cruise, Prochlorococcus ranged between <10(3) and >3 x 10(5) cells ml(-1), Synechococcus between <100 and >1.0 x 10(4) cells ml(-1), and picoeukaryotes between <100 and >10(4) cells ml(-1).
Two empirical models were used to learn more about the relationship between heterotrophic bacteria and nanoflagellates. Most bacterial production was ingested when this production was low, the heterotrophic nanoflagellates could be controlled by preys during Latitud-I cruise at the DCM, and by predators in the surface and in the Latitud-II cruise. Our results were placed in context with others about the structure and function of auto- and heterotrophic picoplankton and heterotrophic nanoplankton in the Central Atlantic Ocean. (C) 2008 Elsevier Ltd. All rights reserved,


Velzeboer I, Hendriks AJ, Ragas AM, Van de Meent D (2008) Aquatic ecotoxicity tests of some nanomaterials. Environ Toxicol Chem 27 :1942-1947

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19086210

Nanoparticles of TiO2, ZrO2, AL2O3, CeO2, fullerene (C60), single-walled carbon nanotubes, and polymethylmethacrylate were tested for ecotoxic effects using one or more ecotoxicity endpoints : Microtox (bacteria), pulse-amplitude modulation (algae), Chydotox (crustaceans), and Biolog (soil enzymes). No appreciable effects were observed at nominal concentrations of up to 100 mg/L. Dilution of nanoparticle suspensions, either in ultrapure (Milli-Q) water or in natural (pond) water, led to formation of larger particles, which settled easily. (Nano)particles in water were characterized by means of atomic force microscopy, energy-dispersive x-ray analysis, inductively coupled plasma-mass spectrometry, flow cytometry, and spectrophotometry. It is concluded that the absence of ecotoxicity is the result of low concentrations of free nanoparticles in the tests, and it is suggested that colloid (in)stability is of primary importance in explaining ecotoxic effects of nanoparticles in the natural environment.


Velzeboer I, Hendriks AJ, Ragas AMJ, Van de Meent D (2008) Aquatic ecotoxicity tests of some nanomaterials. Environmental Toxicology and Chemistry 27 :1942-1947

<Go to ISI> ://000258325000014

Nanoparticles of TiO2, ZrO2, Al2O3, CeO2, fullerene (C-60), single-walled carbon nanotubes, and polymethylmethacrylate were tested for ecotoxic effects using one or more ecotoxicity endpoints : Microtox((R)) (bacteria), pulse-amplitude modulation (algae), Chydotox (crustaceans), and Biolog((R)) (soil enzymes). No appreciable effects were observed at nominal concentrations of up to 100 mg/L. Dilution of nanoparticle suspensions, either in ultrapure (Milli-Q((R))) water or in natural (pond) water, led to formation of larger particles, which settled easily. (Nano) particles in water were characterized by means of atomic force microscopy, energydispersive x-ray analysis, inductively coupled plasma - mass spectrometry, flow cytometry, and spectrophotometry. It is concluded that the absence of ecotoxicity is the result of low concentrations of free nanoparticles in the tests, and it is suggested that colloid (in) stability is of primary importance in explaining ecotoxic effects of nanoparticles in the natural environment.


Vieira RP, Gonzalez AM, Cardoso AM, Oliveira DN, Albano RM, Clementino MM, Martins OB, Paranhos R (2008) Relationships between bacterial diversity and environmental variables in a tropical marine environment, Rio de Janeiro. Environmental Microbiology 10 :189-199

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This study is the first to apply a comparative analysis of environmental chemistry, microbiological parameters and bacterioplankton 16S rRNA clone libraries from different areas of a 50 km transect along a trophic gradient in the tropical Guanabara Bay ecosystem. Higher bacterial diversity was found in the coastal area, whereas lower richness was observed in the more polluted inner bay water. The significance of differences between clone libraries was examined with LIBSHUFF statistics. Paired reciprocal comparisons indicated that each of the libraries differs significantly from the others, and this is in agreement with direct interpretation of the phylogenetic tree. Furthermore, correspondence analyses showed that some taxa are related to specific abiotic, trophic and microbiological parameters in Guanabara Bay estuarine system.


Vilicic D, Terzic S, Ahel M, Buric Z, Jasprica N, Caric M, Mihalic KC, Olujic G (2008) Phytoplankton abundance and pigment biomarkers in the oligotrophic, eastern Adriatic estuary. Environmental Monitoring and Assessment 142 :199-218

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Phytoplankton distribution and environmental characteristics were determined in a shallow, highly stratified and oligotrophic estuary (Zrmanja, eastern Adriatic). Samples were collected in two contrasting seasons ; winter (February 2000), when river discharge was high, and in summer (July 2003), a period of drought. Phytoplankton distribution was closely related to salinity gradients, nutrient levels, and water residence time. Microscopic analysis revealed that phytoplankton was composed mainly of marine diatoms, dinoflagellates, cryptophytes, green flagellates, and coccolithophorids. The dominant biomarker pigments were fucoxanthin, alloxanthin and 19’-hexanoyloxyfucoxanthin, while lower, but indicative contributions of peridinin and chlorophyll b were also noted. Maximum abundance and biomass were found in the middle estuary in winter and in the upper estuary in summer. The estuary is mostly P-limited. Development of chain-forming marine diatoms was evident in winter. Due to the reduced nutrient input in summer, the biomass accumulated in the upper estuary (1,000 ng chlorophyll a l(-1)) was composed mostly of nanoplanktonic unicellular diatoms, nanoplanktonic marine dinoflagellates, cryptophytes, and chlorophytes. The concentrations of about 200 ng l(-1) hex-fuco, suggested that the contribution of prymnesiophytes to total biomass was comparable to that of diatoms and dinoflagellates. In the middle estuary and coastal sea, PO4 and TIN were 3.5 times lower, resulting in a fivefold decrease in biomass (< 100 ng chlorophyll a l(-1)).The oligotrophic Zrmanja and other karstic rivers discharging in the eastern Adriatic Sea, provide insufficient source of nutrients and low productivity of the eastern Adriatic Sea.


Vital M, Hammes F, Egli T (2008) Escherichia coli O157 can grow in natural freshwater at low carbon concentrations. Environ Microbiol 10 :2387-2396

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18507671

Whereas much information on the die-off of Escherichia coli in the aquatic environment is available, only few data support its growth under such conditions. We therefore investigated batch growth in microcosms containing different types of sterile freshwater. The water samples were inoculated with low starting cell concentrations of E. coli O157 (3 x 10(3) cells ml(-1)) and growth was followed using nucleic acid staining combined with flow cytometry. We demonstrated that E. coli O157 is able to grow in sterile freshwater at low carbon concentrations, which is against the common view that cell numbers decline over time when added to freshwater samples. A correlation between apparent assimilable organic carbon (AOC(app)) concentration and the final cell concentration reached by E. coli O157 was established (P < 0.01). A considerable fraction of the AOC(app) (34 +/- 13%) was used by E. coli O157 but the numerical cell yield was about five-times lower in comparison with the bacterial AOC-test community, which originated from natural freshwater. On average, the maximum specific growth rate (mu(max)) of E. coli O157 growing in sterile freshwater at 30 degrees C was 0.19 +/- 0.07 h(-1). Batch growth assays at five different temperatures revealed a positive influence of temperature on mu(max) of E. coli O157. The results give new information on the behaviour of this common pathogen in the aquatic environment and contribute to microbial risk assessment in order to prevent spreading of water-borne diseases.


Wang Y, Hammes F, Duggelin M, Egli T (2008) Influence of size, shape, and flexibility on bacterial passage through micropore membrane filters. Environ Sci Technol 42 :6749-6754

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18800559

Sterilization of fluids by means of microfiltration is commonly applied in research laboratories as well as in pharmaceutical and industrial processes. Sterile micropore filters are subject to microbiological validation, where Brevundimonas diminuta is used as a standard test organism. However, several recent reports on the ubiquitous presence of filterable bacteria in aquatic environments have cast doubt on the accuracy and validity of the standard filter-testing method. Six different bacterial species of various sizes and shapes (Hylemonella gracilis, Escherichia coli, Sphingopyxis alaskensis, Vibrio cholerae, Legionella pneumophila, and B. diminuta) were tested for their filterability through sterile micropore filters. In all cases, the slender spirillum-shaped Hylemonella gracilis cells showed a superior ability to pass through sterile membrane filters. Our results provide solid evidence that the overall shape (including flexibility), instead of biovolume, is the determining factor for the filterability of bacteria, whereas cultivation conditions also play a crucial role. Furthermore, the filtration volume has a more important effect on the passage percentage in comparison with other technical variables tested (including flux and filter material). Based on our findings, we recommend a re-evaluation of the grading system for sterile filters, and suggest that the species Hylemonella should be considered as an alternative filter-testing organism for the quality assessment of micropore filters.


Wang Y, Hammes F, Egli T (2008) The impact of industrial-scale cartridge filtration on the native microbial communities from groundwater. Water Res 42 :4319-4326

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18775553

Groundwater is a major source for bottled water, which is increasingly consumed all over the world. Some categories of bottled water can be subjected to treatments such as disinfection prior to bottling. In the current study, we present the quantitative impact of industrial-scale micro-filtration (0.22 microm pore size) on native microbial communities of groundwater and evaluate subsequent microbial growth after bottling. Two separate groundwater aquifers were tested. Flow-cytometric total cell concentration (TCC) and total adenosine tri-phosphate (ATP) analysis were used to quantify microbial abundance. The TCC of the native microbial community in both aquifers was in the range of 10(3)-10(4) cells/ml. Up to 10% of the native microbial community was able to pass through the cartridge filtration units installed at both aquifers. In addition, all samples (either with or without 0.22 microm filtration) showed significant growth after bottling and storage, reaching average final concentrations of 1-3 x 10(5) cells/ml. However, less growth was observed in carbon-free glassware than in standard polyethylene terephthalate (PET) bottles. Furthermore, our results showed that filtration and bottling can alter the microbial community patterns as observed with flow cytometry. The current study established that industrial-scale micro-filtration cannot serve as an absolute barrier for the native microbial community and provided significant insight to the impact of filtration and bottling on microbial concentrations in bottled water.


Williams CJ, Boyer JN, Jochem FJ (2008) Indirect hurricane effects on resource availability and microbial communities in a subtropical wetland-estuary transition zone. Estuaries and Coasts 31 :204-214

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Three sequential hurricanes made landfall over the South Florida peninsula in August and September 2004. The storm systems passed north of the Everglades wetlands and northeastern Florida Bay, but indirect storm effects associated with changes in freshwater discharge during an otherwise drought year occurred across the wetland-estuary transition area. To assess the impacts of the 2004 hurricane series on hydrology, nutrients, and microbial communities in the Everglades wetlands to Florida Bay transition area, results are presented in the context of a seasonal cycle without hurricane activity (2003). Tropical activity in 2004 increased rainfall over South Florida and the study area, thereby temporarily relieving drought conditions. Not so much actual rainfall levels at the study site but more so water management practices in preparation of the hurricane threats, which include draining of an extensive freshwater canal system into the coastal ocean to mitigate inland flooding, rapidly reversed hypersalinity in the wetlands-estuary study area. Although annual discharge was comparable in both years, freshwater discharge in 2004 occurred predominantly during the late wet season, whereas discharge was distributed evenly over the 2003 wet season. Total organic carbon (TOC), ammonium (NH4+), and soluble reactive phosphorus (SRP) concentrations increased during the hurricane series to concentrations two to five times higher than long-term median concentrations in eastern Florida Bay. Spatiotemporal patterns in these resource enrichments suggest that TOC and SRP originated from the Everglades mangrove ecotone, while NH4+ originated from the bay. Phytoplankton biomass in the bay increased significantly during storm-related freshwater discharge, but declined at the same time in the wetland mangrove ecotone from bloom conditions during the preceding drought. In the bay, these changes were associated with increased nanophytoplankton and decreased picophytoplankton biomass. Heterotrophic bacterial production increased in response to freshwater discharge, whereas bacterial abundance decreased. Hydrochemical and microbial changes were short-lived, and the wetland-bay transition area reverted to more typical oligotrophic conditions within 3 months after the hurricanes. These results suggest that changes in freshwater discharge after drought conditions and during the hurricane series forced the productivity and P-enriched characteristics of the wetland’s mangrove ecotone, although only briefly, to the south into Florida Bay.


Williams CJ, Lavrentyev PJ, Jochem FJ (2008) Bottom-up and top-down control of heterotrophic bacterioplankton growth in a phosphorus-depleted subtropical estuary, Florida Bay, USA. Marine Ecology-Progress Series 372 :7-18

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The influences of resources (carbon, nitrogen, and phosphorus) and protist bacterivory on heterotrophic bacterioplankton growth rates were investigated at 4 sampling sites in the subtropical Florida Bay seagrass estuary during 3 seasons, summer 2004, winter 2005, and fall 2006. Bacterial growth and grazing mortality rates were determined by dilution experiments and flow cytometry, where bacterial communities were separated into high and low DNA content populations (HDNA and LDNA, respectively). In addition, methylumbelliferyl-heptanoate hydrolysis assays were used to measure ambient esterase activity and to determine the impact of resources and grazing on esterase activity. Total bacterial gross growth, grazing mortality, and net growth rates ranged from 0.41 to 1.72, 0.32 to 1.46, and -0.17 to 0.41 d(-1), respectively. HDNA bacteria gross growth and grazing mortality rates were consistently higher than LDNA gross growth and grazing mortality rates. However, LDNA bacteria were not inactive and exhibited positive gross and net growth rates. Stepwise linear multiple regression analysis indicated that In-transformed ammonium concentration and ambient esterase activity related significantly and positively to total bacteria net growth rates (R-2 = 0.75). When total bacterial mortality rates met or exceeded gross growth rates (i.e. negative or zero net growth rates), esterase activity increased with the reduction of grazing pressure without nutrient addition. When bacterial gross growth rates exceeded bacterial grazing mortality rates (i.e. positive net growth rates), both reduction of grazing pressure and nutrient amendments were required to stimulate esterase activity. These results suggest that bottom-up processes regulated heterotrophic bacterial growth, abundance, and carbon use when ammonium concentrations were high and phosphate concentrations were low (i.e. high inorganic N:P ratios). Under low ammonium concentrations and relatively increased phosphate concentrations (i.e. low N:P ratios), top-down processes regulated heterotrophic bacterioplankton growth, abundance, and carbon use in Florida Bay, USA.


Wurl O, Holmes M (2008) The gelatinous nature of the sea-surface microlayer. Marine Chemistry 110 :89-97

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The sea-surface microlayer (SML) represents the interfacial layer between the ocean and atmosphere and covers about 70% of the world’s surface. Gel-like transparent exopolymer particles (TEP) in the SML were studied in oceanic and estuarine SML and subsurface water samples from South East Asia. The TEP enrichment factor, determined as the ratio of the TEP concentration in the SML to that in the corresponding subsurface water, was in the range of 0.39 to 2.43 (1.31 +/- 0.52 mean standard deviation) and 0.29 to 9.72 (1.77 +/- 3.03) in the oceanic and estuarine samples, respectively. Sulfate half-ester groups in the TEP showed a higher enrichment (3.29 +/- 2.36) than the less strongly binding carboxyl groups (1.12 +/- 0.71). Enrichment processes of TEP to the SML are discussed including diffusion to the SML, bubble scavenging and higher production rates of TEP in the SML than in subsurface waters. The results of a general enrichment of gel particles support the concept of a hydrated gelatinous interfacial layer with a complex matrix of dissolved organic matter rather than a more classical model of organized layers of "wet" and "dry" surfactants. (C) 2008 Elsevier B.V. All rights reserved.


Yang Z, Wang W, Liu Y, Kong FX, Zhang M, Shi XL, Cao HS (2008) Increased Growth of Chlorella pyrenoidosa (Chlorophyta) in Response to Substances from the Rotifer Brachionus calyciflorus. Journal of Freshwater Ecology 23 :545-552

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We assessed the response of Chlorella pyrenoidosa to substance(s) from the rotifer Brachionus calyciflorus using flow cytometry and PHYTO-PAM fluorometry. The cell density of C. pyrenoidosa in the rotifer water treatment was significantly higher than that in the control, suggesting that substance(s) from the rotifer stimulated the alga to grow faster. Additionally, the cell size of C. pyrenoidosa in the rotifer water treatment was consistently smaller than that in the control. Esterase activity and chlorophyll fluorescence of C. pyrenoidosa cells in the rotifer water treatment increased and were higher than those in the control but were not significantly different. The maximal efficiency of PSII (Fv/Fm) and the effective quantum yield of PSII (Phi(PSII)) of C. pyrenoidosa in the rotifer water were also consistently higher than those in the control, although these differences also were not significant. The pattern, however, does seem to Support the idea that C. pyrenoidosa responded to infochemical produced by the rotifer.


Yano A, Suzuki K, Yoshizaki G (2008) Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions. Biol Reprod 78 :151-158

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17901070

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.


Yentsch CS, Lapointe BE, Poulton N, Phinney DA (2008) Anatomy of a red tide bloom off the southwest coast of Florida. Harmful Algae 7 :817-826

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A massive outbreak of Karenia brevis that had been ongoing for several months along the southwestern coast of Florida was sampled in early September 2005 off Sanibel Island to assess the utility of bio-optical features and ataxonomic analysis (quantification of eukaryotic and cyanobacterial picoplankton) by flow cytometry in monitoring red tide blooms. Sea-surface sampling followed aircraft visual location of discolored water. Within the most concentrated area of the bloom, chlorophyll a values exceeded 500 mu g l(-1), and concentrations of nitrate (0.3 mu M +/- 0.0) and ammonium (<0.2 mu M) were depleted compared to high concentrations of total dissolved nitrogen, total dissolved phosphorus, and soluble reactive phosphorus (141 +/- 34 mu M, 16.5 +/- 2.5 mu M, and 6.44 +/- 0.57 mu M, respectively). Low water clarity in the bloom (Secchi depth transparency 0.3 m, K-d estimated at 4.83 m(-1)) was strongly influenced by attenuation from dinoflagellates as well as chromophoric dissolved organic matter (CDOM). The fact that the K. brevis bloom occurred in lower-salinity (30 psu), high-nutrient waters implicates riverine transport of land-based nutrients as a source of nutrient supplies that fueled or sustained the bloom. Throughout ongoing efforts to advance modeling and technological capabilities that presently lack reliable predictive capability, bio-optical remote sensing via aerial flyovers along with in-water sensor data can continue to provide accurate coverage of relatively large temporal and spatial features. Flow cytometry can provide conservative (because of some cell lysis), rapid, near-real-time validation of bloom components. The concentration and position of the organisms, along with water mass scalars, can also help to diagnose factors promoting K. brevis bloom development and dispersion. (C) 2008 Elsevier B.V. All rights reserved.


Zhang H, Bhattacharya D, Maranda L, Lin S (2008) Mitochondrial cob and cox1 genes and editing of the corresponding mRNAs in Dinophysis acuminata from Narragansett Bay, with special reference to the phylogenetic position of the genus Dinophysis. Appl Environ Microbiol 74 :1546-1554

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18165361

Dinophysis acuminata cells were isolated from Narragansett Bay water samples in June 2005 using flow cytometry. Dinoflagellate-specific PCR primers were used to isolate small-subunit rRNA (18S rRNA), mitochondrial cytochrome b (cob), and cytochrome c oxidase I (cox1) genes and the encoded cDNAs. Maximum-likelihood analysis of a concatenated data set of ribosomal DNA and cDNA sequences of cob and cox1 showed that D. acuminata was sister to Gonyaulacoids, but without strong bootstrap support. The approximately unbiased test could not reject alternative positions of D. acuminata. To gain better resolution, mRNA editing of cob and cox1 was inferred for D. acuminata and 13 other dinoflagellate species. The location and type of editing as well as the distribution pattern in D. acuminata were generally similar to those in other dinoflagellates except for two edited sites that are unique to this species. Bayesian analyses of a matrix that recorded the location and type of editing, and of a matrix that included the protein sequences of COB and COX1 with the editing data yielded tree topologies similar to the three-gene tree but again failed to resolve the phylogenetic position of D. acuminata. However, the density of edited sites in the D. acuminata mitochondrial genes, consistent with phylogenetic trees, indicated that Dinophysis is a derived dinoflagellate lineage, diverging after other lineages such as Oxyrrhis, Amphidinium, and Symbiodinium. We demonstrate that dinoflagellate-specific PCR coupled with flow cytometry can be a useful tool to analyze genes and their transcripts from a natural dinoflagellate population.


Zubkov MV, Tarran GA, Mary I, Fuchs BM (2008) Differential microbial uptake of dissolved amino acids and amino sugars in surface waters of the Atlantic Ocean. Journal of Plankton Research 30 :211-220

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Nitrogen bioavailability is considered to limit the productivity of oceanic oligotrophic gyres, the largest biomes on Earth. In order to assess the microbial requirement for small organic nitrogen molecules in these and other waters, the microbial uptake rates of amino acids (leucine, methionine and tyrosine) and amino sugars (glucosamine and N-acetyl-glucosamine) as well as glucose were compared using a bioassay technique of radiotracer dilution. The bioassays were carried out on four mid-Atlantic meridional transects spanning a latitudinal range from 60 degrees N to 42 degrees S. The mean concentrations of both bioavailable N-acetyl-glucosamine and glucose in the gyres were 1 nM, four times higher than the mean leucine concentration. Despite its lower concentration, the mean turnover time of leucine in the gyres of 15 h was 90 and 9 times shorter than the turnover time of N-acetyl-glucosamine and glucose, respectively. In addition, among amino acids, leucine was taken up in the gyres at a rate of 1.5 times faster than methionine and 2.5 times faster than tyrosine. Hence, oceanic bacterioplankton as a community showed a clear preference for amino acids, particularly leucine, compared with amino sugars. The preferential uptake of amino acids to sugars challenges the concept of microbial nitrogen or carbon limitation in the open ocean.