mardi 21 avril 2009
par   G. Grégori

Adams MS, Stauber JL (2004) Development of a whole-sediment toxicity test using a benthic marine microalga. Environ Toxicol Chem 23 :1957-1968


An acute whole-sediment toxicity test with a benthic marine microalga was developed and optimized using flow cytometry to distinguish algae (based on their chlorophyll a autofluorescence) from sediment particles. Of seven benthic marine algae screened, the diatom Entomoneis cf punctulata was most suitable because of its tolerance of a wide range of water and sediment physicochemical parameters, including salinity, pH, ammonia, and sulfide. A whole-sediment and water-only toxicity test based on inhibition of esterase activity in this species was developed. Enzyme activity rather than growth was used as the test endpoint, as nutrient release from sediments has previously been found to stimulate algal growth, potentially masking contaminant toxicity. The sensitivity of the bioassay to a range of metals (copper, zinc, cadmium, lead, arsenic, manganese) and phenol in water-only exposures was compared to the standard 72-h growth rate inhibition test. The esterase enzyme inhibition test was sensitive to copper, with a 3-h inhibitory concentration to cause a 50% (IC50) reduction in a fluorescein diacetate fluorescence value of 97 +/- 39 microg Cu/L. A concentration-dependent response was also observed in the presence of sediment particles (copper tailings), with and without dilution, using a control clean sediment. The primary route of exposure to copper was via pore water rather than by direct contact with tailings particles. This is the first whole-sediment bioassay developed with a benthic alga suitable for sediment quality assessment in marine/estuarine systems, and its advantages and limitations are discussed.

Andrade L, Gonzalez AM, Valentin JL, Paranhos R (2004) Bacterial abundance and production in the southwest Atlantic Ocean. Hydrobiologia 511 :103-111

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A bacterial dynamics study was conducted in the southwest Atlantic Ocean off the Brazilian coast from October to December 1998 with samples collected at the depth of chlorophyll a maximum. The bacteria were counted by flow cytometry with the SYTO13 DNA dye and bacterial activity was measured as carbon production by the uptake of H-3-leucine. The samples were also analysed for basic hydrographical variables. Bacterial counts varied from 2.1 to 9.7 x 10(5) cells ml(-1) and bacterial production varied from 4.6 to 126.6 ng C L-1 h(-1). Spatial distribution of nitrate, chlorophyll a, bacterial abundance and bacterial activity showed areas of water fertilization in the oligotrophic Atlantic Ocean. Statistical analysis of data characterized the division of the area in two by latitude 19degreesS. On the north fertilizations are related to river discharge and on the south to the input of nutrients from deep waters. Remobilisation of nutrients in those spots results among all in higher biological activity. However the relation between bacterial abundance and activity is top-down controlled characterizing most of the waters as oligotrophic. Eddies showed by bacterial data can probably sustain the fisheries resources for the whole area. The influence of gradients across eddies on biological activity and food chain structure should be an important question to be studied in oligotrophic oceans.

Boldyrev A, Bulygina E, Leinsoo T, Petrushanko I, Tsubone S, Abe H (2004) Protection of neuronal cells against reactive oxygen species by carnosine and related compounds. Comp Biochem Physiol B Biochem Mol Biol 137 :81-88


Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.

Borghese R, Borsetti F, Foladori P, Ziglio G, Zannoni D (2004) Effects of the metalloid oxyanion tellurite (TeO32-) on growth characteristics of the phototrophic bacterium Rhodobacter capsulatus. Appl Environ Microbiol 70 :6595-6602


This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 microg of tellurite (TeO3(2-)) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 microg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (< or =50% viable cells) ; however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (> or =90% viable cells), which was supported by the development of a significant membrane potential (Deltapsi = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.

Bouvy M, Troussellier M, Got P, Arfi R (2004) Bacterioplankton responses to bottom-up and top-down controls in a West African reservoir (Selingue, Mali). Aquatic Microbial Ecology 34 :301-307

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We conducted experiments to determine whether bacterial growth, with or without predators, is limited by inorganic (N and P) and organic (C) substrates in Selingue, a mesotrophic reservoir located in Mali, West Africa. Significant increases (relative to controls) in bacterial cell volumes and thymidine incorporation rates were observed after 24 h incubation only for samples amended with the combination +PC and +CNP. The data revealed a colimitation of bacterioplankton growth by organic carbon during the dry season. Flow cytometry discriminated 3 groups of bacteria (Bact I, Bact II, Bact III) differing in increases in nucleic acid content and cell size. The Bact I group, comprising cells with low nucleic acid content and of small size, was the dominant population in all experiments. In the absence of bacterial predators, only the Bact II group showed significant differences between the control and the +PC and +CNP treatments, indicating that this bacterial group was the most sensitive to nutrient additions. The Bact II group, corresponding to cells with high DNA content, are active members of the bacterioplankton community. The Bact III group did not increase in any treatment but the proportions of these cells always increased in the presence of bacterial predators. These cells may be, at least partly, grazing - resistant bacteria.

Briand E, Pringault O, Jacquet S, Torreton JP (2004) The use of oxygen microprobes to measure bacterial respiration for determining bacterioplankton growth efficiency. Limnology and Oceanography-Methods 2 :406-416

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Heterotrophic bacterial growth efficiency (BGE), the ratio between the carbon consumed and the bacterial biomass produced, is a key factor in understanding flows of organic matter in aquatic ecosystems. Methods generally used to estimate bacterial respiration require long incubations ( 24 to 36 h) to measure significant rates during which nonlinear patterns of oxygen decrease may bias BGE computation. These respiration estimates are generally compared to bacterial production rates determined from radiotracer incorporation from much shorter incubations. The aim of this study was to improve the determination of bacterial respiration to better estimate BGE. For this purpose, we employed oxygen microprobes in predator free ( 0.6 mum filtered) seawater samples and determined in parallel bacterial abundance. The use of oxygen microprobes allowed us to continuously monitor oxygen concentration during the incubation. Hence, the length of incubation can be adjusted as soon as a significant decrease of oxygen is observed. At the most productive sites, respiration was measurable from the beginning of the incubation and varied with time. In contrast, at the oligotrophic sites, respiration was often detectable only after a lag-phase of 5 to 10 h and remained constant thereafter. BGE was computed from the changes in bacterial abundance observed during the respiration measurements. This way, both processes were determined in similar incubation conditions. In comparison, the use of radiotracer derived bacterial production systematically resulted in an underestimation of BGE.

Brussaard CP (2004) Optimization of procedures for counting viruses by flow cytometry. Appl Environ Microbiol 70 :1506-1513


The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Brussaard CPD (2004) Optimization of procedures for counting viruses by flow cytometry. Applied and Environmental Microbiology 70 :1506-1513

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The development of sensitive nucleic acid stains, in combination with How cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in How cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80degreesC. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 X 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80degreesC, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80degreesC) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Brussaard CPD, Short SM, Frederickson CM, Suttle CA (2004) Isolation and phylogenetic analysis of novel viruses infecting the phytoplankton Phaeocystis globosa (Prymnesiophyceae). Applied and Environmental Microbiology 70 :3700-3705

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Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.

Button DK, Robertson B, Gustafson E, Zhao XM (2004) Experimental and theoretical bases of specific affinity, a cytoarchitecture-based formulation of nutrient collection proposed to supercede the Michaels-Menten paradigm of microbial kinetics. Applied and Environmental Microbiology 70 :5511-5521

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A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells(-1) h(-1) found were up to 10(3) times smaller than those from a "Marinobacter arcticus" isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well ; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K-m was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K-m as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.

Caballero S, Abad FX, Loisy F, Le Guyader FS, Cohen J, Pinto RM, Bosch A (2004) Rotavirus virus-like particles as surrogates in environmental persistence and inactivation studies. Appl Environ Microbiol 70 :3904-3909


Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20 degrees C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20 degrees C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

Calvo-Diaz A, Moran XAG, Nogueira E, Bode A, Varela M (2004) Picoplankton community structure along the northern Iberian continental margin in late winter-early spring. Journal of Plankton Research 26 :1069-1081

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The surface distribution of autotrophic and heterotrophic picoplankton was assessed in 24 transects perpendicular to the coast along the N and NW Iberian peninsula shelf in late winter and early spring 2002. Community structure was analyzed by flow cytometry (FC) and found to be strongly influenced by hydrography. Typical late winter conditions were found during the survey, characterized by the presence of the poleward Portugal coastal counter current (PCCC) in the west and an increasing stratification eastwards. Cyanobacteria (mostly Synechococcus) dominated at low chlorophyll a (Chl a) concentration whereas both the total and relative abundance of picoeukaryotes generally increased with total phytoplankton biomass. Differences in the cell size of most FC-defined picoplanktonic groups were also observed along the longitudinal and coastal-offshore gradients. The presence of Prochlorococcus (<10(3) cells mL(-1)) coincided with the core of the PCCC and its significant correlation with salinity suggests its possible use as a tracer of this current. Two groups of heterotrophic bacteria were distinguished according to their relative DNA content. High DNA bacteria dominated the community (60 +/- 1% SE of total numbers), reaching maximum values in areas under riverine influence with presumed higher inputs of organic matter. Picoplankton biomass was dominated by heterotrophic bacteria in the western region (58 +/- 3%) while autotrophic groups contributed on average 66 +/- 2% in the southern Bay of Biscay. The heterotrophic bacteria to phytoplankton biomass ratio decreased significantly along the measured range. Yet showing regional differences, the estimated contribution of picophytoplankton to total algal biomass was high (mean 59 +/- 4%), indicating the important role of small cells at the onset of the spring bloom in these temperate shelf waters.

Cao A, Ramos-Martinez JI, Barcia R (2004) In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk. Fish Shellfish Immunol 16 :215-225


The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.

Cardenas W, Dankert JR, Jenkins JA (2004) Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides. Fish & Shellfish Immunology 17 :223-233

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Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Re treated cells (P less than or equal to 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertebrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds. (C) 2004 Elsevier Ltd. All rights reserved.

De Guise S, Levin MJ (2004) Cetacean-reconstituted severe combined immunodeficient (SCID) mice respond to vaccination with canine distemper vaccine. Vet Immunol Immunopathol 97 :177-186


Morbillivirus infections have been responsible for mass mortalities in several species of marine mammals. Nevertheless, relatively little is known on the pathogenesis of the disease and the immune response to the agent, especially in cetaceans, hindering the treatment of individuals and the development of appropriate vaccines, given the difficulty of performing experimental work in marine mammals. The reconstitution of severe combined immunodeficient (SCID) mice, which do not have the ability to reject grafts, with lymphocytes from different species has been used with increasing success as a surrogate species model to study the immune system. We injected NOD/SCID mice with lymphocytes from different species of cetaceans and further vaccinated those mice with a commercial canine distemper virus (CDV) vaccine to develop a practical model to study cetacean immune response to a morbillivirus. Reconstitution was detected in 10/20 mice reconstituted with harbor porpoise spleen, 6/10 mice reconstituted with harbor porpoise lymph node cells, 8/10 mice reconstituted with fresh beluga PBMCs and none of the mice reconstituted with neonate bottlenose dolphin spleen or thymus cells when assessed 42-63 days after reconstitution. While a humoral immune response was detected in none of the reconstituted mice, a cell-mediated immune response to the CDV vaccine was detected in 6/15 (40%) and 2/18 (11%) of the SCID mice after reconstitution with cetacean immune cells after a single or booster vaccination, respectively, for a combined total of 8/33 (24%). This represents the first demonstration of successful reconstitution of SCID mice with marine mammal cells, and to the authors’ knowledge, the first direct demonstration of a primary antigen-specific cell-mediated immune response in reconstituted SCID mice. This model will be useful for further research on the physiology of the marine mammal immune system and its response to infectious agents and vaccines, with possible important outcomes in conservation issues.

Dooner M, Cerny J, Colvin G, Demers D, Pimentel J, Greer D, Abedi M, McAuliffe C, Quesenberry P (2004) Homing and conversion of murine hematopoietic stem cells to lung. Blood Cells Molecules and Diseases 32 :47-51

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The hematopoietic stem cell population, lineage negative-Sca positive (HSC), displays a homing defect into bone marrow (BM) after 48-h exposure to interleukin (IL)-3, IL-6, IL-11, and steel factor [J. Hematother. Stem Cell Res. 11 (2002) 913]. Cytokine treatment of marine marrow leads to reversible alterations in adhesion protein expression, which may explain the changes in homing. We evaluated 3 It homing to northematopoictic organs of marrow cells exposed to cytokines for 0, 18, 24, 40 and 48 h. HSC cells from C57BL/6J mice were cultured and labeled with the cytoplasmic fluorescent dye CFSE. We found homed events from uncultured cells in spleen, liver and lung, but no events were seen in duodenum or anterior tibialis muscle. Culture in cytokines led to decreased homing to marrow at 24 and 48 It with parallel changes in spleen homing. There was little variability of homing to liver, however the number, of homed events in lung was markedly increased when 24-h cultured cells were assessed. This was approximately a 10-fold increase compared to the 0 h time point (flow cytometry). Homing was determined by evaluation of frozen section (8 pm) by fluorescent microscopy for spleen, liver, duodenum, anterior tibialis and lung. Data were confirmed by flow cytometry from each organ including marrow. These data indicate the presence of a lung homing "hotspot" at 24 h of cytokine culture ; this is a time when the stem progenitors cells are in mid S-phase. Altogether these data suggest that homing of marrow cell to nonmarrow organs may fluctuate with cell cycle transit and that there is a lung homing hotspot in mid-S. (C) 2003 Elsevier Inc. All rights reserved.

Dubelaar GBJ, Geerders PJF (2004) Innovative technologies to monitor plankton dynamics - Scanning flow cytometry : a new dimension in real-time, in-situ water quality monitoring. Sea Technology 45 :15-+

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Phytoplankton is an important water-quality indicator due to its high species differentiation, fast growth rates and responsiveness to environmental actuators. National and regional regulations and directives call for a detailed assessment of phytoplankton blooms as an indicator of the ecological status of various types of waters. Microscopic analysis of samples takes a lot of time and, therefore, only detects low-frequency changes. Automated systems, such as scanning cytometry, can fill the gaps between microscopical examinations by gathering information on rapid, high-frequency changes and, thus, permit real-time, in-situ operational monitoring. This article discusses modern scanning flow cytometry as a practical means to obtain real-time information on changes in plankton communities in marine, coastal and estuarine waters.

Dubelaar GBJ, Geerders PJF, Jonker RR (2004) High frequency monitoring reveals phytoplankton dynamics. Journal of Environmental Monitoring 6 :946-952

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Phytoplankton is an important water quality indicator because of its high species differentiation, growth rates and responsiveness to environmental actuators. The new European Water Framework Directive calls for assessment of the duration, intensity and succession of phytoplankton blooms to determine the ecological status of various types of waters. For common phytoplankton growth rates basic signal processing theory yields a minimum monitoring frequency of once per day, which is much more than applied in standard practice. To assess the nature of this discrepancy we followed the behaviour of about 40 groups of organisms/particles found in the Oude Rijn river by a two-week daily cytometric analysis. Particle counts of the 20 most abundant groups are shown. Their variation rate and magnitude confirm that daily sampling is needed to follow such ecosystems in detail. It is shown that limiting the monitoring to the "coarse line’’ does not allow a correspondingly decreased sampling frequency. Automated systems may fill the gaps between the microscopical examinations by gathering highly frequent information. The information depth of bulk measurements is poor however, and not used as such. The data shown here demonstrate that modern scanning flow cytometry (SFC) offers an information depth close to the taxonomic level. In the past decade, acquisition and operation costs of these systems have come down considerably, whereas operation is hands free, even in situ and submerged, and data analysis has become more efficient. SFC is used most efficiently complementary to microscopical analyses for mutual validation. In these cases it presents a realistic solution to generate the essential high frequency observations required to assess ecosystem variability.

Eldridge ML, Trick CG, Alm MB, DiTullio GR, Rue EL, Bruland KW, Hutchins DA, Wilhelm SW (2004) Phytoplankton community response to a manipulation of bioavailable iron in HNLC waters of the subtropical Pacific Ocean. Aquatic Microbial Ecology 35 :79-91

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Studies in high nutrient, low chlorophyll (HNLC) regions have demonstrated that increased Fe availability results in an increase in phytoplankton biomass and changes in community composition. Here we present experiments in which the availability of iron (Fe) was increased or reduced to monitor the response of individual groups of phytoplankton (large eukaryotes, picoeukaryotes and cyanobacteria) by flow cytometry, Additions (0.5 to 5.0 nM Fe) and reductions in available Fe (through addition of 1 to 10 nM of the fungal siderophore desferrioxamine B) were made to enclosed communities from the South American eastern boundary current off Peru, where ambient dissolved Fe concentrations were <100 pM. As predicted, chlorophyll concentrations increased in the added Fe treatments relative to the control, indicative of Fe limitation. Flow cytometry demonstrated that this was due to increases in the abundance of large eukaryotes that are Fe-starved under ambient conditions. Cyanobacterial abundance increased and decreased linearly with Fe availability, suggesting that cyanobacteria were Fe-limited but not Fe-starved. In contrast, picoeukaryote cell abundance increased with decreasing Fe availability, although chlorophyll cell(-1) in this group responded in an inverse manner. The results demonstrate that members of the marine phytoplankton community respond differently to Fe availability, which may influence the outcome of biological competition among organisms in Fe-limited environments.

Estrada M, Henriksen P, Gasol JM, Casamayor EO, Pedros-Alio C (2004) Diversity of planktonic photo auto trophic microorganisms along a salinity gradient as depicted by microscopy, flow cytometry, pigment analysis and DNA-based methods. Fems Microbiology Ecology 49 :281-293

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The diversity of prokaryotic and eukaryotic phytoplankton was studied along a gradient of salinity in the solar salterns of Bras del Port in Santa Pola (Alacant, Spain) using different community descriptors. Chlorophyll a, HPLC pigment composition, flow cytometrically-determined picoplankton concentration, taxonomic composition of phytoplanktori (based on optical microscopy) and genetic fingerprint patterns of 16S (cyanobacteria- and chloroplast-specific primers) and 18S rRNA genes were determined for samples from ponds with salinities ranging from 4% to 37% Both morphological and genetical descriptors of taxonomic composition showed a good agreement and indicated a major discontinuity at salinities between 15% and 22%. The number of classes and the Shannon diversity index corresponding to the different descriptors showed a consistent decreasing trend with increasing salinity. The results indicate a selective effect of extremely high salinities on phytoplanktonic assemblages. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Faria JMR, van Lammeren AAM, Hilhorst HWM (2004) Desiccation sensitivity and cell cycle aspects in seeds of Inga vera subsp affinis. Seed Science Research 14 :165-178

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The desiccation sensitivity of seeds of Inga vera Willd. subsp. affinis, a recalcitrant-seeded tree from Brazil, was analysed, focusing on water relations and cell-cycle aspects, including DNA content and the microtubular cytoskeleton. Seeds were collected at four developmental stages, dried to different moisture contents (MCs), assessed regarding water activity and set to germinate. Samples of fresh (non-dried) developing and mature seeds were used for assessment of DNA content by flow cytometry. Immunohistochemical detection of microtubules (MTs) was done in mature seeds at different MCs. Slight desiccation of immature seeds increased germination, but further drying resulted in a quick decline of germinability. During seed development the desiccation sensitivity decreased slightly, but DNA content of the embryonic axis cells remained constant, suggesting no relation between those two parameters. Embryonic axis cells of mature seeds showed abundant cortical microtubule arrays, which were not affected by mild desiccation, but broken down by further drying. It appeared that, upon rehydration, damaged cells were not able to reconstitute the microtubular cytoskeleton. The failure of germination of Inga vera seeds after drying could not be attributed to cellular damage to DNA synthesis and mitosis, since the radicle protruded by means of cell elongation, without a need for cell division. However, the breakdown of MTs during desiccation, and their subsequent inability to reassemble upon rehydration, may be related to the decreased germination, since MTs are required for cell elongation.

Ferri LE, Pascual J, Seely AJE, Giannias B, Christou NV (2004) Intra-abdominal sepsis attenuates local inflammation-mediated increases in microvascular permeability at remote sites in mice in vivo. Surgery 135 :187-195

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Background. Given that leukocyte delivery to remote sites is diminished in states of systemic inflammation, such as sepsis, and activated leukocytes may be responsible for endothelial injury leading to vascular leakage, we hypothesized that intra-abdominal sepsis would diminish microvascular leakage at remote sites by altering leukocyte-endothelial interactions.
Methods. Using a marine intravital microscopy model, we examined leukocyte-endothelial interactions and vascular leakage at a peripheral site in the presence of local and/or systemic inflammation. Forty mice were randomized to 1 of 4 study groups : local infection (orchitis), systemic infection (intra-abdominal sepsis by cecal ligation and puncture), local and systemic infection, and control. Postcapillary venules of the cremaster muscle were examined by bright light and fluorescence intravital microscopy. Microvascular leakage was determined after intravenous administration of fluorescent albumin.
Results. Systemic infection attenuated the increases in both leukocyte adherence and local infection-induced microvascular permeability. Neutrophil cell-surface expression of L-selectin, as determined by flow cytometry, diminished with both local and systemic infection, whereas expression of CD11b increased with systemic, but not local, infection.
Conclusions. These data suggest that systemic (intra-abdominal) sepsis diminishes local inflammation-mediated vascular leakage by attenuating leukocyte adherence.

Fouilland E, Descolas-Gros C, Courties C, Collos Y, Vaquer A, Gasc A (2004) Productivity and growth of a natural population of the smallest free-living eukaryote under nitrogen deficiency and sufficiency. Microb Ecol 48 :103-110


The influence of dissolved inorganic nitrogen (DIN) enrichments on cell-normalized carbon uptake rate, chlorophyll a content, and apparent cell size of a picoeukaryote (<1 microm) ( Ostreococcus tauri, the smallest eukaryotic cell) from a natural summer phytoplanktonic assemblage (<200 microm) in a northern Mediterranean Lagoon (Thau Lagoon) was studied in 20-L enclosures in June 1995. The natural planktonic community was incubated in situ for 24 h with initial ammonium and nitrate enrichments and compared to a control without enrichment. O. tauri cell-normalized productivity was estimated from the combination of flow cytometric (FCM) enumeration and 2-h (radioactive) carbonate incorporation measured on post-incubation size fractions (<1microm). No difference between the effects of the two DIN sources of enrichment on the studied biological parameters was measured during this experiment. Growth of natural O. tauri was perturbed by the low DIN availability in the control with drastic changes in cell productivity, chlorophyll content, and cell cycle (from the variations in apparent cell size) as compared to the DIN sufficiency conditions. On the other hand, a very high specific growth rate for natural O. tauri, up to 8 day(-1) under DIN enrichments, has been estimated from production and abundance data obtained during this experiment. This supports values measured in culture and suggests that the yearly high contribution of picophytoplankton to the total primary production in Thau Lagoon is likely to be due to their high growth rate rather than the previously suggested lack of grazing pressure.

Franklin NM, Stauber JL, Lim RP (2004) Development of multispecies algal bioassays using flow cytometry. Environ Toxicol Chem 23 :1452-1462


Multispecies algal bioassays, suitable for assessing copper toxicity, were developed with three marine (Micromonas pusilla, Phaeodactylum tricornutum, and Heterocapsa niei) and three freshwater (Microcystis aeruginosa, Pseudokirchneriella subcapitata, and Trachelomonas sp.) microalgae. Flow cytometry was used to separate and count algal signals based on pigment fluorescence and cell size. Species were mixed together on the basis of equivalent surface areas to avoid the confounding effect on toxicity of increased biomass for metal binding. Under control conditions (no added copper), M. pusilla growth was inhibited in the presence of the other marine microalgae compared to single-species tests, while the opposite was true (i.e., growth stimulation) for M. aeruginosa and P. subcapitata in freshwater mixtures. Competition for nutrients, including CO2, and algal exudate production may account for these effects. Interactions between microalgal species also had a significant effect on copper toxicity to some species. In freshwater multispecies bioassays, the toxicity of copper to Trachelomonas sp. was greater in the presence of other species, with copper concentrations required to inhibit growth (cell division) rate by 50% (72-h [IC50]) decreasing from 9.8 to 2.8 microg Cu/L in single- and multispecies bioassays, respectively. In contrast, in marine multispecies bioassays, copper toxicity to the marine diatom P. tricornutum was reduced compared to single-species bioassays, with an increase in the 72-h IC50 value from 13 to 24 microg Cu/L. This reduction in copper toxicity was not explained by differences in the copper complexing capacity in solution (as a result of exudate production) because labile copper, measured by anodic stripping voltammetry, was similar for P. tricornutum alone and in the mixture. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.

Fukuda H, Koike I (2004) Microbial stimulation of the aggregation process between submicron-sized particles and suspended particles in coastal waters. Aquatic Microbial Ecology 37 :63-73

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Transfer rates of submicron-sized particles (SMP) to micro-suspended particles (5 to 20 mum in diameter) in coastal waters around Japan were measured using fluorescent beads (0.5 mum in diameter) and flow cytometry, with and without metabolic inhibitors (thiuram. or NaN3) This flow cytometric technique has the advantage that it allows the number of beads stuck to suspended particles to be counted without any pre-treatment, such as filtration, that can introduce artifacts. The transfer rates to particles in the 5-20 mum fraction without inhibitors were on average 4.3 to 5.8 times higher than with added NaN3. On the other hand, ingestion rates of fluorescent beads by protozoa in the 5-20 mum fraction were less than 10% of the transfer rates to particles in the 5-20 mum fraction. These results indicate that biological activities were more efficient than physical mechanisms for the transfer process of SMP into the 5-20 mum fraction separately from the grazing process. Also, the inhibitory effect of thiuram on the transfer rates suggests an involvement of eukaryotic cells in this stimulation. The intensity of this stimulation was positively correlated with the density of attached nanoflagellates per particle (Da) (R = 0.630, n = 22), bacterial abundance (R = 0.489, n = 22), concentration of chlorophyll a (R = 0.518, n = 18) and water temperature (R = 0.569, n = 22). These results indicate that biological processes are key mechanisms for the coagulation process of submicro- and micro-scale particles in coastal environments.

Gagnaire B, Thomas-Guyon H, Renault T (2004) In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes. Fish Shellfish Immunol 16 :501-512


In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.

Gasol JM, Casamayor EO, Joint I, Garde K, Gustavson K, Benlloch S, Diez B, Schauer M, Massana R, Pedrs-Alio C (2004) Control of heterotrophic prokaryotic abundance and growth rate in hypersaline planktonic environments. Aquatic Microbial Ecology 34 :193-206

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The factors controlling prokaryote abundance and activity along salinity gradients were investigated in the Bras del Port solar saltern system (Alacant, Spain) in May 1999. Specific growth rates were high and prokaryote abundance relatively low at the lowest (seawater) salinities ; the opposite was found at higher salinities. Experiments were carried out in representative salterns at salinities of 4 to 37%, to test whether prokaryote abundance and growth rate were (1) limited by inorganic or organic nutrients (nutrient addition experiments), (2) limited by cell abundance (dilution experiments), or (3) affected by zooplankton cascading down to affect the prokaryote predators. Low-salinity ponds were limited by organic nutrients, while high-salinity ponds responded slightly only to dilution. Zooplankton affected prokaryote growth rates particularly in the medium-salinity ponds. In the low salinity ponds, zooplankton effects were weak and probably indirect (through increased supply of organic matter). Neither organic matter limitation nor zooplankton predation pressure affected prokaryote development in the higher salinity ponds. We suggest that 3 types of functional communities occur in the same saltern system : (1) an active, substrate-limited community in the low salinity ponds ; (2) an active, grazer-controlled community in the medium salinity ponds ; and (3) a possibly dormant, probably substrate-limited, community in the high salinity ponds. However, the results at the highest salinities were equivocal, because the dilution manipulation had detrimental effects, artificially decreasing the contribution of the haloarchaea, which were essential contributors to the total activity in the saltern. Bacterial taxonomic community composition was also determined in these experiments by denaturing gradient gel electrophoresis (DGGE) analyses on 16S rRNA genes, and showed very small changes in community composition in the experimental manipulations. Together with the known microbial community structure and composition at differing salinities along the gradient, our results show that functional aspects of the microbial food web also vary between salterns.

Havskum H, Schluter L, Scharek R, Berdalet E, Jacquet S (2004) Routine quantification of phytoplankton groups - microscopy or pigment analyses ? Marine Ecology-Progress Series 273 :31-42

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Phytoplankton pigments in samples taken from nutrient-enriched and non-enriched 3 m(3) seawater enclosures were separated and quantified using high-performance liquid chromatography (HPLC). The enclosures were with and without inorganic (N, P, Si) and organic (glucose, C) nutrient enrichments, resulting in a variation of phytoplankton groups in time and space. The relative contribution of the major phytoplankton groups to the total chlorophyll a (i.e. chlorophyll a plus chlorophyllide a) was estimated by the CHEMTAX program. The results were compared to phytoplankton groups identified and quantified by light and epifluorescence microscopy. For the pigmented flagellate groups the results obtained by microscopy and pigment analyses using the CHEMTAX program showed similar trends. The picocyanobacteria were readily quantified by microscopy and the results were similar to those obtained by flow-cytometry, while the CHEMTAX program for the cyanobacteria revealed different trends. Microscopy and pigment analyses provided similar trends in diatom population development. Estimated diatom contributions to total phytoplankton biomass, however, were considerably higher when based on microscopy than when based on the CHEMTAX program, especially in Si-amended enclosures. Total chlorophyll a:carbon ratios for diatoms were at the lower end of a previously reported range between 1:27 and 1:67. For the pigmented flagellate groups the total chlorophyll a:carbon ratios were above that range. In routine monitoring of phytoplankton we recommend the use of the CHEMTAX program based on HPLC pigment analyses accompanied by a screening for the dominating species by microscopy, and by flow-cytometry for quantification of picocyanobacteria.

Hawley TS, Herbert DJ, Eaker SS, Hawley RG (2004) Multiparameter flow cytometry of fluorescent protein reporters. Methods Mol Biol 263 :219-238


Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed). The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm. Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.

Hirano T, Yamauchi N, Sato F, Soh T, Hattori MA (2004) Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes. J Reprod Dev 50 :599-603


Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes : a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.

Jang HN, Woo JK, Cho YH, Kyong SB, Choi SH (2004) Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin. J Biochem Mol Biol 37 :314-319


Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system.

Jochem FJ, Lavrentyev PJ, First MR (2004) Growth and grazing rates of bacteria groups with different apparent DNA content in the Gulf of Mexico. Marine Biology 145 :1213-1225

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Growth rates and grazing losses of bacterioplankton were assessed by serial dilution experiments in surface waters in the Mississippi River plume, the northern Gulf of Mexico, a Texas coastal lagoon (Laguna Madre), southeast Gulf of Mexico surface water, and the chlorophyll subsurface maximum layer in the southeast Gulf of Mexico. Bacteria were quantified by flow cytometry after DNA staining with SYBR Green, which allowed for discrimination of growth and grazing rates of four bacteria subpopulations distinguished by their apparent DNA content and cell size ( light scatter signal). Total bacteria growth rates (0.2 - 0.9 day(-1)) were mostly balanced by grazing losses, resulting in net growth rates of - 0.18 to 0.45 day(-1). Growth rates of DNA subpopulations varied within experiments, sometimes substantially. In most, but not all, experiments, the largest bacteria with highest DNA content exhibited the highest growth rates, but a relationship between DNA content and growth rates or grazing losses was absent. Small bacteria with the lowest DNA content showed positive growth rates in most experiments, sometimes higher than growth rates of bacteria containing more DNA, and were grazed upon actively. Low- DNA bacteria were not inactive and were an integral part of the microbial food web.

Johnson P, Lebaron P, Deromedi T, Baudart J, Catala P (2004) Tests of a fountain flow cytometry system for real-time quantification of rare microorganisms in an aquatic environment. Cytometry Part A 59A :104-104

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Kobayashi T, Yoshizaki G, Takeuchi Y, Takeuchi T (2004) Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry. Mol Reprod Dev 67 :91-100


A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67 : 91-100, 2004.

Larsen A, Flaten GAF, Sandaa RA, Castberg T, Thyrhaug R, Erga SR, Jacquet S, Bratbak G (2004) Spring phytoplankton bloom dynamics in Norwegian coastal waters : Microbial community succession and diversity. Limnology and Oceanography 49 :180-190

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Most studies of spring bloom succession in Norwegian waters have employed light microscopy and accounted for species composition of phyto- and zooplankton. Flow cytometry and molecular tools enable us to extend such investigations to include smaller organisms like bacterio- and virioplankton. Here, we describe succession and diversity of algae, bacteria, and viruses in relation to environmental changes from 15 February to 27 April. The spring succession started with an increase in autotrophic picoeukaryotes and Synechococcus sp. The diatoms bloomed around the middle of March and caused nutrient depletion in the upper part of the water column. Upwelling in the beginning of April gave rise to a second bloom, consisting of diatoms and Phaeocystis pouchetii. Numerically, autotrophic picoeukaryotes and Synechococcus sp. dominated the periods between and after these two major blooms. Heterotrophic bacterial abundance increased throughout the experimental period and reached peak values during and after phytoplankton blooms. These bacteria were succeeded by viruses having low DNA fluorescence, whereas viruses with medium DNA fluorescence bloomed during or after blooms of autotrophic picoeukaryotes. High-DNA fluorescence viruses reached maximum concentrations during and after the diatom and Phaeocystis blooms. The diversity of the bacterial community remained relatively stable, whereas viral diversity varied more and increased after major phytoplankton blooms. Our investigation thus demonstrates how virioplankton are important elements of the total microbial diversity and how they are intimately linked to the rest of the microbial community and possibly act as an internal driving force in spring bloom successions.

Lavrentyev PJ, McCarthy MJ, Klarer DM, Jochem F, Gardner WS (2004) Estuarine microbial food web patterns in a Lake Erie coastal wetland. Microb Ecol 48 :567-577


Composition and distribution of planktonic protists were examined relative to microbial food web dynamics (growth, grazing, and nitrogen cycling rates) at the Old Woman Creek (OWC) National Estuarine Research Reserve during an episodic storm event in July 2003. More than 150 protistan taxa were identified based on morphology. Species richness and microbial biomass measured via microscopy and flow cytometry increased along a stream-lake (Lake Erie) transect and peaked at the confluence. Water column ammonium (NH4+) uptake (0.06 to 1.82 microM N h(-1)) and regeneration (0.04 to 0.55 microM N h(-1)) rates, measured using 15NH4+ isotope dilution, followed the same pattern. Large light/dark NH4+ uptake differences were observed in the hypereutrophic OWC interior, but not at the phosphorus-limited Lake Erie site, reflecting the microbial community structural shift from net autotrophic to net heterotrophic. Despite this shift, microbial grazers (mostly choreotrich ciliates, taxon-specific growth rates up to 2.9 d(-1)) controlled nanophytoplankton and bacteria at all sites by consuming 76 to 110% and 56 to 97% of their daily production, respectively, in dilution experiments. Overall, distribution patterns and dynamics of microbial communities in OWC resemble those in marine estuaries, where plankton productivity increases along the river-sea gradient and reaches its maximum at the confluence.

Levin M, Morsey B, Mori C, Guise S (2004) Specific non-coplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure. J Toxicol Environ Health A 67 :1517-1535


Contaminant-induced immunosuppression by organochlorines (OC), particularly polychlorinated biphenyls (PCBs), has been suspected as a cofactor in the deaths of thousands of marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study was aimed at characterizing the immunomodulatory potential of representative OCs on phagocytosis in bottlenose dolphins and beluga whales. The ability of peripheral blood leukocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar PCB congeners, 138, 153, and 180, one coplanar PCB, 169, and 2,3,7,8-TCDD and all possible mixtures (26) were tested upon in vitro exposure. In both species, all mixtures containing at least two non-coplanar PCBs significantly reduced both neutrophil and monocyte phagocytosis, with effects more marked in dolphins than in belugas. Coplanar OCs, on their own or when added to non-coplanar congeners, did not further modulate phagocytosis, suggesting an Ah receptor-independent mechanism. Concentration-response experiments with individual congeners further demonstrated a non-coplanar PCB-induced suppression of phagocytosis, while coplanar congeners produced no consistent effects. Our results suggest simple additive interactions of chemicals in a mixture. However, calculation of toxic equivalency (TEQs) failed to predict the experimentally induced immunomodulatory effects of OCs on dolphin and beluga phagocytosis, confirming the Ah receptor-independent nature of the effects on phagocytosis. Overall, our results suggest that non-AhR mechanisms may explain one facet of immunotoxicity (phagocytosis), something that is not captured using the TEQ approach. This is the first report demonstrating the immunomodulatory effects of OCs on dolphin and beluga phagocytosis, and the first overall demonstration of immunomodulatory effects on phagocytosis mediated specifically by non-coplanar PCBs.

Lezcano M, Granja C, Salazar M (2004) The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei). Cryobiology 48 :349-356


Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.

Lin SJ, Mulholland MR, Zhang H, Feinstein TN, Jochem FJ, Carpenter EJ (2004) Intense grazing and prey-dependent growth of Pfiesteria piscicida (Dinophyceae). Journal of Phycology 40 :1062-1073

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Grazing and growth of Pfiesteria piscicida (Pfiest) were investigated using batch and cyclostat cultures with Rhodomonas sp. (Rhod) as prey. Observed maximum growth rates (1.4 d(-1)) and population densities (2 x 10(5) cells.mL(-1)) corresponded to values predicted by Monod functions (1.76 d(-1) ; 1.4 x 10(5) cells.mL(-1)). In batch cultures under a range of prey-to-predator ratios (0.1:1 to 180:1) and prey concentrations (1000-71,000 cells.mL(-1)), Rhodomonas sp. was always depleted rapidly and P. piscicida concentrations increased briefly. The rate of Rhodomonas sp. depletion and the magnitude of P. piscicida population maxima depended on the prey-to-predator ratio and prey concentration. Starvation resulted in cell cycle arrest at G1 and G2+M and ultimately the demise of both P. piscicida and Rhodomonas sp. populations, demonstrating the dependence of P. piscicida on the supply of appropriate prey. The depletion of Rhodomonas sp. populations could be attributed directly to grazing, because P. piscicida did not exert detectable inhibitory effects on the growth of Rhodomonas sp. but grazed intensely, with maximum grazing rates>10 Rhod.Pfiest(-1).d(-1) and with no apparent threshold prey abundance for grazing. The results suggest that 1) the abundance of appropriate prey may be an important factor regulating P. piscicida abundance in nature, 2) P. piscicida may control prey population, and 3) high growth and grazing potentials of P. piscicida along with cell cycle arrest may confer survival advantages.

Lisle JT, Priscu JC (2004) The occurrence of lysogenic bacteria and microbial aggregates in the lakes of the McMurdo Dry Valleys, Antarctica. Microbial Ecology 47 :427-439

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The McMurdo Dry Valleys of Antarctica form the coldest and driest ecosystem on Earth. Within this region there arc a number of perennially ice-covered (3-6 m thick) lakes that support active microbial assemblages and have a paucity of metazoans. These lakes receive limited allochthonous input of carbon and nutrients, and primary productivity is limited to only 6 months per year owing to an absence of sunlight during the austral winters. In an effort to establish the role that bacteria and their associated viruses play in carbon and nutrient cycling in these lakes, indigenous bacteria, free bacteriophage, and lysogen abundances were determined. Total bacterial abundances (TDC) ranged from 3.80 x 10(4) to 2.58 x 10(7) cells mL(-1) and virus-like particle (VLP) abundances ranged from 2.26 x 10(5) to 5.56 x 10(7) VLP mL(-1). VLP abundances were significantly correlated (P < 0.05) with TDC, bacterial productivity (TdR), chlorophyll a (Chl a), and soluble reactive phosphorus (SRP). Lysogenic bacteria, determined by induction with mitomycin C, made up between 2.0% and 62.5% of the total population of bacteria when using significant decreases and increases in TDC and VLP abundances, respectively, and 89.5% when using increases in VLP abundances as the sole criterion for a successful induction event. The contribution of viruses released from induced lysogens contributed <0.015% to the total viral production rate. Carbohydrate and protein based organic aggregates were abundant within the water column of the lakes and were heavily colonized by bacteria and VLPs. Alkaline phosphatase activity was detected within the matrix of the aggregates, implying phosphorus deficiency and consortial nutrient exchanges among microorganisms.

Matsumoto K, Furuya K, Kawano T (2004) Association of picophytoplankton distribution with ENSO events in the equatorial Pacific between 145 degrees E and 160 degrees W. Deep-Sea Research Part I-Oceanographic Research Papers 51 :1851-1871

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Climatological variability of picophytoplankton populations that consisted of >64% of total chlorophyll a concentrations was investigated in the equatorial Pacific. Flow cytometric analysis was conducted along the equator between 145degreesE and 160degreesW during three cruises in November-December 1999, January 2001, and January-February 2002. Those cruises were covering the La Nina (1999, 2001) and the pre-El Nino (2002) periods. According to the sea surface temperature (SST) and nitrate concentrations in the surface water, three regions were distinguished spatially, viz., the warm-water region with > 28 degreesC SST and nitrate depletion (< 0.1 mumol kg(-1)), the upwelling region with < 28 degreesC SST and high nitrate (>4 mumol kg(-1)) water, and the in-between frontal zone with low nitrate (0.1-4 mumol kg(-1)). Picophytoplankton identified as the groups of Prochlorococcus, Synechococcus and picocukaryotes showed a distinct spatial heterogeneity in abundance corresponding to the watermass distribution. Prochlorococcus was most abundant in the warm-water region, especially in the nitrate-depleted water with > 150 x 10(3) cells ml(-1), Synechococcus in the frontal zone with > 15 x 10(3) cells ml(-1), and picoeukaryotes in the upwelling region with > 8 x 10(3) cells ml(-1). The warm-water region extended eastward with eastward shift of the frontal zone and the upwelling region during the pre-El Nino period. On the contrary, these regions distributed westward during the La Nina period. These climatological fluctuations of the watermass significantly influenced the distribution of picophytoplankton populations. The most abundant area of Prochlorococcus and Synechococcus extended eastward and picoeukaryotes developed westward during the pre-El Nino period. The spatial heterogeneity of each picophytoplankton group is discussed here in association with spatial variations in nitrate supply, ambient ammonium concentration, and light field. (C) 2004 Elsevier Ltd. All rights reserved.

Miao B, Geng M, Li J, Li F, Chen H, Guan H, Ding J (2004) Sulfated polymannuroguluronate, a novel anti-acquired immune deficiency syndrome (AIDS) drug candidate, targeting CD4 in lymphocytes. Biochem Pharmacol 68 :641-649


Sulfated polymannuroguluronate (SPMG), a marine sulfated polysaccharide, has entered the Phase II clinical trial in China as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate obtained from marine organisms. To determine the binding site(s) (receptors) of SPMG in lymphocytes mediating its anti-AIDS activities, fluorescein-5-isothiocyanate (FITC)-labeled SPMG was used to investigate SPMG binding to lymphocytes. Flow cytometry (FCM) and fluorescence microscopy analysis showed that the SPMG binds to lymphocytes in a rapid, specific, reversible, and saturable fashion. Several SPMG binding proteins were purified by affinity chromatography from lymphocyte membrane preparations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis revealed that a 55kDa lymphocyte membrane protein is CD4. To characterize the SPMG and CD4 interaction, inhibition assay and surface plasmon resonance (SPR) assay were carried out. SPMG bound to CD4 in a multivalent fashion with specificity. The binding of SPMG to human lymphocyte CD4 was competitively inhibited by human soluble CD4 (hsCD4). Likewise, the binding between hsCD4 and immobilized SPMG was blocked by excess free SPMG. These results indicate that CD4 is one of the specific SPMG binding sites (receptors) in lymphocytes. The interaction between SPMG and CD4 may provide a mechanistic explanation of the immunopotentiating and anti-AIDS activities of SPMG in human immunodeficiency virus (HIV) infected individuals.

Miller JH, Rouwe B, Gaitanos TN, Hood KA, Crume KP, Backstrom BT, La Flamme AC, Berridge MV, Northcote PT (2004) Peloruside A enhances apoptosis in H-ras-transformed cells and is cytotoxic to proliferating T cells. Apoptosis 9 :785-796


Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells ; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.

Minor EC, Nallathamby PS (2004) "Cellular" vs. "detrital" POM : a preliminary study using fluorescent stains, flow cytometry, and mass spectrometry. Marine Chemistry 92 :9-21

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The mass of nonliving particulate organic matter (POM) in marine waters can be significantly greater than that in living cells, yet very little is known about the composition and ultimate fate of this "detrital" POM. In recent marine chemistry studies, flow cytometry has been used to provide further insights into the composition, source, and fate of chlorophyll-fluorescing POM (which includes living algal cells) vs. detrital POM within natural waters. In this study, we extend such morphological characterization of POM beyond the use of chlorophyll autofluorescence and particle size or light scatter. Instead, nucleic acid staining with SYTO-13 and flow cytometry sorting were used to identify and isolate "cellular’ and "detrital’ subclasses of POM from samples collected in the Lafayette River and the Elizabeth River (subestuaries in the lower Chesapeake Bay). The resulting subclasses, as well as aliquots of unsorted POM sample, were then analyzed by direct temperature-resolved mass spectrometry. We show that "cellular" and "detrital" subclasses differ significantly from each other, and that detrital POM appears chemically distinct from the bulk POM. We then suggest further applications for flow cytometry approaches in aquatic organic biogeochemistry. (C) 2004 Elsevier B.V. All rights reserved.

Miyadai T, Ootani M, Tahara D, Aoki M, Saitoh K (2004) Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes). Fish Shellfish Immunol 17 :211-222


Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.

Moreno F, Laine L (2004) The flow cytometry, a new tool for trophic coastal ecosystem diagnostic. Ecological Indicators 4 :161-176

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The aim of this study is to develop a synthetic ecological index from phytoplanktonic data.
An ecological index was developed from 78 flow cytometric data sampled in four sites (3 in Madagascar and I in Mozambique) and 108 samples (2 malagasy and 1 moroccan sites) for validation. This index describes the trophic level, the microbial loop activity and the stability of the system.
According to several coastal phytoplanktonic ecological studies, this index defines the ecological status of the micro-ecosystem studied in 62.96% of the cases.
The validation, made with lagoonal Mediterranean data, allows to extend the application area from the inter-tropical zone to the Mediterranean coasts. (C) 2004 Elsevier Ltd. All rights reserved.

Murrel MC, Lores EM (2004) Phytoplankton and zooplankton seasonal dynamics in a subtropical estuary : importance of cyanobacteria. Journal of Plankton Research 26 :371-382

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A seasonal study of phytoplankton and zooplankton was conducted from 1999 to 2001 in Pensacola Bay, Florida, USA, to further the understanding of pelagic food webs in sub-tropical estuaries. Monthly measurements included size-fractionated chlorophyll (whole water, <5 mum, <20 mum), net- and picophytoplankton composition analyzed using microscopy, flow cytometry, and HPLC pigment analysis. Additionally, zooplankton abundance and dry weight were determined from net tows. The results show a phytoplankton community dominated by the small size fraction (<5 mum), especially during the warm periods. The <5> 5-10 mum) were predominantly composed of diatoms, followed by chlorophytes, cryptophytes and dinoflagellates. The three most abundant genera of diatoms were Thalassiosira, Pennales and Cyclotella. Zooplankton biomass averaged 12.2 mug C L-1, with peak biomass occurring during May (similar to30 mug C L-1). Zooplankton abundance averaged 16.7 ind. L-1, peaking at 30 ind. L-1 during May. During the summer, the zooplankton community shifted from the ubiquitous Acartia tonsa towards Oithona sp. The increase in Oithona coincided with increases in picophytoplankton and may reflect the changing food resources available to zooplankton. Thus, the trophic implications of cyanobacterial dominance in sub-tropical estuaries need to be more fully assessed.

Orellana MV, Petersen TW, van den Engh G (2004) UV-excited blue autofluorescence of Pseudo-nitzschia multiseries (Bacillariophyceae). Journal of Phycology 40 :705-710

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A flow cytometer coupled to a scanning monochromator and a fluorescence microscope were used to characterize the fluorescence spectrum of Pseudo-nitzschia multiseries (Haste) Haste, a pennate diatom that produces the neurotoxin domoic acid, a lethal amnesic. In this research, we characterize the fluorescence spectrum of P multiseries in vivo over the wavelength range of 360 to 850 nm and show that this diatom autofluoresces blue when excited with UV light (350-365 nm). The autofluorescence characterization of Pseudo-nitzschia may provide new methods for rapid in situ monitoring of diatom populations and reiterates the usefulness of flow cytometry in the analysis and study of marine phytoplankton.

Pelegrin P, Chaves-Pozo E, Mulero V, Meseguer J (2004) Production and mechanism of secretion of interleukin-1beta from the marine fish gilthead seabream. Dev Comp Immunol 28 :229-237


Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.

Peters AF, Marie D, Scornet D, Kloareg B, Cock JM (2004) Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism for brown algal genetics and genomics. Journal of Phycology 40 :1079-1088

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The emergence of model organisms that permit the application of a powerful combination of genomic and genetic approaches has been a major factor underlying the advances that have been made in the past decade in dissecting the molecular basis of a wide range of biological processes. However, the phylogenetic distance separating marine macroalgae from these model organisms, which are mostly from the animal, fungi, and higher plant lineages, limits the latters’ applicability to problems specific to macroalgal biology. There is therefore a pressing need to develop similar models for the macroalgae. Here we describe a survey of potential model brown algae in which particular attention was paid to characteristics associated with a strong potential for genomic and genetic analysis, such as a small nuclear genome size, sexuality, and a short life cycle. Flow cytometry of nuclei isolated from zoids showed that species from the Ectocarpales possess smaller haploid genomes (127-290 Mbp) than current models among the Laminariales (580-720 Mbp) and Fucales (1095-1271 Mbp). Species of the Ectocarpales may complete their life histories in as little as 6 weeks in laboratory culture and are amenable to genetic analyses. Based on this study, we propose Ectocarpus siliculosus (Dillwyn) Lyngbye as an optimal choice for a general model organism for the molecular genetics of the brown algae.

Pires LMD, Jonker RR, Van Donk E, Laanbroek HJ (2004) Selective grazing by adults and larvae of the zebra mussel (Dreissena polymorpha) : application of flow cytometry to natural seston. Freshwater Biology 49 :116-126

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1. Selective grazing of adults and larvae of the zebra mussel (Dreissena polymorpha) on phytoplankton and detritus from both laboratory cultures and natural seston was quantified using flow cytometry.
2. Mean clearance rate of adult zebra mussels was higher on a mixture of the green alga Scenedesmus and the cyanobacterium Microcystis than when Scenedesmus was offered as single food, suggesting selective feeding by the mussels.
3. Feeding on lake seston both adults and larvae showed a higher clearance rate on phytoplankton than on detritus particles, suggesting that zebra mussels select for phytoplankton. Furthermore, it was noted that adults preferred seston particles in the 0-1 and 30-100 mum size ranges.
4. In our study, zebra mussels did not discriminate against cyanobacteria, and our results indicate that they may even ingest them preferentially.

Porter J, Morris SA, Pickup RW (2004) Effect of trophic status on the culturability and activity of bacteria from a range of lakes in the English Lake District. Applied and Environmental Microbiology 70 :2072-2078

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The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolitum salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.

Readman JW, Devilla RA, Tarran G, Llewellyn CA, Fileman TW, Easton A, Burkill PH, Mantoura RF (2004) Flow cytometry and pigment analyses as tools to investigate the toxicity of herbicides to natural phytoplankton communities. Mar Environ Res 58 :353-358


Characterisation of natural phytoplanktonic communities is currently being advanced through flow cytometry and high resolution pigment analyses. To date, toxicological methods to assess impacts of herbicides on natural phytoplankton populations are lacking. Here, we report the novel use of these techniques in combination to study changes in phytoplankton populations exposed to 2-methylthio-4-tertiary-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051), a herbicide used in antifouling paints. Flow cytometry results revealed that following a 72-h exposure to approximately 100 ngL(-1), eukaryote abundance was less than half that in the controls. High performance liquid chromatographic analyses of pigments demonstrated that 19’-hexanoyloxyfucoxanthin was selectively lost relative to the control. This carotenoid is specific to the prymnesiophytes which are key constituents of phytoplanktonic communities within temperate marine waters. Values of EC(50) (72 h) as low as 70 ngL(-1) were calculated from the selective reduction in this compound. Concentrations substantially exceeding this level have been reported in UK and other European coastal waters.

Readman JW, Devilla RA, Tarran G, Llewellyn CA, Fileman TW, Easton A, Burkill PH, Mantoura RFC (2004) Flow cytometry and pigment analyses as tools to investigate the toxicity of herbicides to natural phytoplankton communities. Marine Environmental Research 58 :353-358

<Go to ISI> ://000222199100038

Characterisation of natural phytoplanktonic communities is currently being advanced through flow cytometry and high resolution pigment analyses. To date, toxicological methods to assess impacts of herbicides on natural phytoplankton populations are lacking. Here, we report the novel use of these techniques in combination to study changes in phytoplankton populations exposed to 2-methylthio-4-tertiary-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051((R))), a herbicide used in antifouling paints. Flow cytometry results revealed that following a 72-h exposure to approximately 100 ng L-1, eukaryote abundance was less than half that in the controls. High performance liquid chromatographic analyses of pigments demonstrated that 19’-hexanoyloxyfucoxanthin was selectively lost relative to the control. This carotenoid is specific to the prymnesiophytes which are key constituents of phytoplanktonic communities within temperate marine waters. Values of EC50 (72 h) as low as 70 ng L-1 were calculated from the selective reduction in this compound. Concentrations substantially exceeding this level have been reported in UK and other European coastal waters. (C) 2004 Published by Elsevier Ltd.

Regel RH, Brookes JD, Ganf GG, Griffiths R (2004) The influence of experimentally generated turbulence on the Mash01 unicellular Microcystis aeruginosa strain. Hydrobiologia 517 :107-120

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Phytoplankton experience a continuously changing fluid environment and the response to this is reflected at individual and community levels. The large-scale motions of winds, waves and artificial circulations are coupled by turbulence to the viscous small-scale environment of the phytoplankton cell. To investigate the significance of turbulence in the ecology of Microcystis aeruginosa, cultures were exposed to turbulent conditions using a vertically oscillating grid for a period of 7 days under controlled laboratory conditions. M. aeruginosa was exposed to a range of turbulent intensities, by adjusting the frequency of oscillation from 1 to 4 Hz. To improve the resolution of scale between turbulence phenomena and phytoplankton, flow cytometry and fluorescent probes were used to assess the response of M. aeruginosa. Metabolic activity and cell viability were monitored daily in both the turbulent cultures and quiescent control cultures using the FDA and Sytox green fluorescent probes, respectively. Initially, low turbulence levels generated by the grid at frequencies of 1 and 2 Hz stimulated metabolic activity, and did not affect cell viability compared to the control quiescent cultures. However, higher levels of turbulence generated by the grid at frequencies of 3 and 4 Hz were deleterious to metabolic activity and viability. Metabolic activity significantly decreased and over 85% of cells were nonviable after 96 h at a grid oscillation of 4 Hz. It was concluded that due to the long lag time (> 96 h) and high intensities needed to exert a deleterious effect, small-scale turbulence is unlikely to be a significant factor controlling M. aeruginosa compared to large scale motion which lead to changes in light and nutrient conditions.

Rose JM, Caron DA, Sieracki ME, Poulton N (2004) Counting heterotrophic nanoplanktonic protists in cultures and aquatic communities by flow cytometry. Aquatic Microbial Ecology 34 :263-277

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The food vacuole stain LysoTracker Green(R) was used to enumerate heterotrophic protists on a standard model flow cytometer. Appropriate stain concentration and staining time were determined using cultures of protists. Stained heterotrophic protists consistently formed distinct populations within cytograms of green fluorescence versus forward scatter. Cytometric counts of cultured species were compared to direct counts using light microscopy at cell abundances ranging from 10(3) to 10(6) cells ml(-1). A regression of these data was highly significant and yielded a slope of 0.95. Stained populations were accurately counted during lag, exponential and early stationary growth phases. Growth rates calculated from cytometric counts were not statistically different from those based on microscopy. The method was applied to 26 natural plankton samples, and general region definitions on the cytograms were established that identified heterotrophic protistan assemblages. A regression of cytometric counts versus direct counts yielded a slope of 1.16. LysoTracker Greene(R) can only be used with live samples because preservation destroys membrane potential, resulting in loss of fluorescence. However, the flow cytometric method employing LysoTracker Greene is highly applicable for monitoring the growth of many heterotrophic protists in cultures and has the potential to be extremely useful for field samples, providing comparable counts to microscopical methods while allowing much faster sample processing.

Russo J, Lagadic L (2004) Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environmental Pollution 127 :303-311

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Immunotoxicological effects of environmentally relevant concentrations (10, 23, 50, 100 mug/l) of atrazine were studied in Lyllinaeal stagnalis. Individual hemolymph sampling was performed at 0, 24, 48, 72, 96, 168, 336, 504 and 672 h during exposure. Every atrazine concentration induced a significant increase in the mean number of circulating hemocytes, without any concentration-response relation. A peak (1.6-fold increase) of hemocyte density was observed after 96 h of exposure. After 504 h, the number of hemocytes remained hi her only in the snails exposed to the two highest concentrations. Granulocytes contributed most to the increase in hemocyte density in herbicide-exposed snails. Both short- (24 and 96 h) and long-term (504 h) exposures resulted in significant inhibition of hemocyte phagocytic activity upon E. coli. Over the long-term, phagocytosis recovered for the two lowest concentrations. After 504 h of exposure, every herbicide level resulted in a significant reduction of reactive oxygen species production in E. coli-stimulated hemocytes, which was not observed for short-term exposures. (C) 2003 Elsevier Ltd. All rights reserved.

Sato F, Nakagawa T, Ito M, Kitagawa Y, Hattori MA (2004) Application of RNA interference to chicken embryos using small interfering RNA. J Exp Zoolog A Comp Exp Biol 301 :820-827


Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Schwartz JA, Aldridge BM, Stott JL, Mohr FC (2004) Immunophenotypic and functional effects of bunker C fuel oil on the immune system of American mink (Mustela vison). Vet Immunol Immunopathol 101 :179-190


The relationship between exposure to environmental contaminants and immunotoxicity in vulnerable marine species is unknown. In this study, we used American mink (Mustela vision) as a surrogate species for the sea otter to examine the immunotoxic effects of chronic exposure to a low concentration of bunker C fuel oil (500 ppm admixed in the feed for 113-118 days). The mink immune system was monitored over time by flow cytometric analysis for alterations in the immunophenotype of blood lymphocytes and monocytes and by mitogen-stimulated proliferation assays for changes in peripheral blood mononuclear cell function. Fuel oil exposure caused a mild, yet significant (P < 0.05) increase in the absolute numbers of specific peripheral blood lymphocyte subsets (CD3+T cells) and monocytes, an increase in the level of expression of functionally significant cell surface proteins (MHC II, CD18), and an increase in mitogen-induced mononuclear cell proliferative responses. This heightened state of cellular activation along with the increase in specific cell surface protein expression on both the innate and adaptive immune cells is similar to the pro-inflammatory or "adjuvant-like" effect described in laboratory models of polycyclic aromatic hydrocarbon exposure in other species. These results show the benefits of using a controlled laboratory model for detecting and characterizing subtle petroleum oil-induced perturbations in immune responses. In addition this study establishes a framework for studying the effects of environmental petroleum oil exposure on the immune system of free-ranging marine mammals. Expansion of these studies to address biolgical significance is warranted.

Sekar R, Fuchs BM, Amann R, Pernthaler J (2004) Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Appl Environ Microbiol 70 :6210-6219


We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

Seymour JR, Mitchell JG, Seuront L (2004) Microscale heterogeneity in the activity of coastal bacterioplankton communities. Aquatic Microbial Ecology 35 :1-16

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Microscale sampling techniques and flow cytometry were employed to measure the distribution patterns of 8 subpopulations of bacteria separated according to variations in cell fluorescence and light scatter properties. Subpopulations of bacteria defined on the basis of these parameters have recently been shown to represent cells exhibiting dissimilar activity levels, and we therefore assume that the subpopulations of bacteria identified here represent metabolically diverse groups. Microscale distribution patterns of these subpopulations were measured at a resolution of 4.5 and 12 mm, within 2 dissimilar coastal habitats, A mean 2-fold change in the abundance of the total bacterial community across sample sets was observed. However, levels of spatial heterogeneity were consistently higher for the cytometrically defined subpopulations than total counts. In most samples, the population of bacteria exhibiting the highest levels of green fluorescence, or DNA content, and hence assumed to represent the most active bacteria in the community, also showed the highest levels of microscale spatial variability, with a maximum change in abundance of 14.5-fold observed across a distance of 9 nun. Where Zipf rank-size analysis was conducted, the microscale distributions of subpopulations differed significantly (p < 0.05) in 79% of cases, implying that bacterial communities are made up of physiologically distinct compartments, perhaps influenced by different microscale features of the environment. We suggest that these results provide the first evidence for the existence of microscale heterogeneity in the metabolic activity of aquatic bacterial communities.

Sipkema D, Snijders AP, Schroen CG, Osinga R, Wijffels RH (2004) The life and death of sponge cells. Biotechnol Bioeng 85 :239-247


Cell viability is an essential touchstone in the study of the effect of medium components on cell physiology. We developed a flow-cytometric assay to determine sponge-cell viability, based on the combined use of fluorescein diacetate (FDA) and propidium iodide (PI). Cell fluorescence measurements based on incubation of cells with FDA or PI resulted in a useful and reproducible estimate of the viability of primary sponge-cell cultures. We studied the effects of temperature, ammonium, and the fungicide amphotericin B on the viability of a primary-cell culture from the marine sponge Suberites domuncula using the aforementioned flow-cytometric assay. S. domuncula cells die rapidly at a temperature of >or=22 degrees C, but they are insensitive to ammonium concentrations of up to 25 mM. Amphotericin B, which is frequently used in sponge-cell culture media, was found to be toxic to S. domuncula cells.

Six C, Thomas JC, Brahamsha B, Lemoine Y, Partensky F (2004) Photophysiology of the marine cyanobacterium Synechococcus sp. WH8102, a new model organism. Aquatic Microbial Ecology 35 :17-29

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Synechococcus spp. constitute a major and ubiquitous component of marine ecosystems. The genome of one strain of this genus, WH8102, has recently been completely sequenced. Since it can also be genetically manipulated, this clone has the potential to become a new model organism however, to date, it remains poorly characterised in terms of pigment composition, optical properties and photophysiology. It has a very high phycourobilin to phycoerythrobilin (PUB:PEB) ratio (ca. 1,95 at low light), and is therefore representative of Synechococcus populations found in oligotrophic areas of the ocean. We show here that this strain has a very wide growth irradiance range from < 15 to > 650 mumol photons m(-2) s(-1) continuous white light, with a maximum growth rate (mu(max) = 1.13 +/- 0.02 d(-1)) at 207 mumol quanta m(-2) s(-1) (I-max). As cells acclimated to high light, drastic variations in the chlorophyll a (chl a), beta-carotene and phycoerythrin (PE) contents were observed, reaching a quasi steady state around In contrast, the zeaxanthin content remained approximately constant whatever the light level. Similarly, the carbon and nitrogen contents did not significantly vary with irradiance, Red and orange fluorescences, as measured by flow cytometry, were found to correlate well with chl a and PE contents, respectively. Spectrometric analyses of phycobilisome (PBS)-containing fractions from cells grown under different photon fluxes suggest. a specific reduction of the PEII content relative to other phycobiliproteins (PBPs) during acclimation of the PBSs to high light.

Sobrino C, Montero O, Lubian LM (2004) UV-B radiation increases cell permeability and damages nitrogen incorporation mechanisms in Nannochloropsis gaditana. Aquatic Sciences 66 :421-429

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This study shows the response of Nannochloropsis gaditana, a marine nannoplanktonic species, exposed to UV radiation for 7 days. PAR, UV-A and UV-B ratios used were within the range likely to be observed in nature, a photoperiod of 12L : 12D was maintained, and light irradiances were modified daily to promote cell acclimation. Growth, pigment content, internal nitrogen and carbon content, and photochemical efficiency using PAM fluorometry were assessed in nutrient replete cultures. Cell size, autofluorescence and cell permeability were analysed by flow cytometry. Results showed a cessation of growth after day 3 and a progressive decrease was observed in F-v/F-m values in cultures exposed to UV-B ( plus UV-A and PAR). Flow cytometry analysis also demonstrated an increase in membrane permeability caused by UV-B damage. Cells that showed an increase in membrane permeability also exhibited a proportional decrease in cellular nitrogen content. The results support the conclusion that UV-B radiation can affect N. gaditana nitrogen incorporation mechanisms by direct damage or indirectly by damage to membrane structure and to the photosynthetic apparatus with resulting effects on energy and reductant demand. In contrast, the presence of UV-A radiation was beneficial to cells exposed to PAR plus UVA when compared to those exposed to only-PAR from day 4. This response resulted in cells with a higher nitrogen content and without changes in membrane permeability.

Steinhagen D, Helmus T, Maurer S, Michael RD, Leibold W, Scharsack JP, Skouras A, Schuberth HJ (2004) Effect of hexavalent carcinogenic chromium on carp Cyprinus carpio immune cells. Dis Aquat Organ 62 :155-161


Chromium is widely used in industrial processes, and is released into aquatic environments by electroplating, tannery and textile industries. Fishes in natural waters or in aquaculture facilities supplied with these waters are exposed to chromium waste and are presumed to be affected by deposits. Herein, we examine the effect of hexavalent chromium on carp Cyprinus carpio derived immune cells. In vitro exposure of carp leukocytes to hexavalent chromium induced cytotoxicity, decreased mitogen-induced lymphocyte activation and phagocyte functions at concentrations between 2 and 200 micromol Cr l(-1). Neutrophils responded to chromium challenge by changes in cell shape together with reduced nitric oxide and reactive oxygen production. This occurred at much lower concentrations than for the cytotoxic effects seen in leukocyte cultures derived from peripheral blood or pronephros. In a similar way, activation of carp lymphocytes by pokeweed mitogen was reduced in a dose-dependent manner, while cytotoxic effects on non-activated lymphocytes were observed at much higher doses of 200 micromol Cr l(-1). Altered lymphocyte and neutrophil functions are considered to be responsible for decreased resistance to pathogens observed in fishes under chronic chromium challenge.

Taira H, Aoki S, Yamanoha B, Taguchi S (2004) Daily variation in cellular content of UV-absorbing compounds mycosporine-like amino acids in the marine dinoflagellate Scrippsiella sweeneyae. Journal of Photochemistry and Photobiology B-Biology 75 :145-155

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Sudden exposure experiments to high PAR (photosynthetically available radiation) or high PAR + UVR (ultraviolet radiation) were conducted for the marine dinoflagellate Scrippsiella sweeneyae acclimated to either low PAR or high PAR to determine the induction of cellular mycosporine-like amino acid (MAA) in relation to photosynthesis status. When the exposure to high PAR (30.8 Wm(-2)) was provided at different time in the light period for S. sweeneyae acclimated to low PAR (7.7 Wm(-2)) which suppressed photosynthesis, S. sweeneyae could enhance the induction of MAA but it only occurred in the first half of the light period. When UVR exposure was provided for the culture acclimated to high PAR which enhanced photosynthesis, cellular MAA content did not increase during the entire light period, but displayed daily variation similar to the control for two and half days. Daily variation of cellular MAA content did not synchronized with that of cell volume and cellular chlorophyll a content. The individual MAAs also revealed similar daily variations with different phase, which increased for a few hours in the beginning of the light period, except for cellular palythine content. Thus the total cellular MAA content revealed daily variation with changing the relative composition within a few hours. As one of the biological protective strategies against harmful UVR in sunlight, the daily vertical migration in the bloom forming dinoflagellates might be accompanied by the daily variation of cellular MAA content for a photosynthesis at daytime. (C) 2004 Elsevier B.V. All rights reserved.

van der Wolf JM, van Beckhoven JRCM (2004) Factors affecting survival of Clavibacter michiganensis subsp sepedonicus in water. Journal of Phytopathology 152 :161-168

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The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non-sterile surface water at 10degreesC, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non-mucoid strain was also studied in sterile ditch water and simulated ’drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20degreesC, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4degreesC do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non-culturable state.

Vaulot D, Le Gall F, Marie D, Guillou L, Partensky F (2004) The Roscoff Culture Collection (RCC) : a collection dedicated to marine picoplankton. Nova Hedwigia 79 :49-70

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The Roscoff Culture Collection (RCC, http://www.sb-roscoff.fr/Phyto/RCC/index.php) holds over 500 strains of marine cyanobacteria and microalgae with a strong emphasis on picoplankton, i.e. cells with size below 2-3 microns. Most of these strains have been obtained from the Equatorial Pacific, the Tropical Atlantic, the Mediterranean Sea, the Red Sea, the North Sea and the English Channel. A large fraction of strains are characterized based on ultrastructure features, pigment analyses and SSU rDNA sequencing. With respect to prokaryotes, the RCC holds more than 100 strains of Prochlorococcus and Synechococcus. Both genera exhibit considerable ecotypic variability since they colonize virtually all oceanic waters from tropical oligotrophic regions to temperate coastal areas. These two cyanobacteria should receive increasing attention with the recent availability of complete genome sequences of four representative isolates. In recent years, we have also isolated novel picoeukaryotic strains from several algal classes (Prasinophyceae, Prymnesiophyceae, Bolidophyceac, Dictyochophyceae, Pelagophyceae...). As an example, we have now 20 strains of Ostreococcus (Prasinophyceae, Mamiellales), the smallest photosynthetic eukaryote initially isolated from Thau lagoon in France. These Ostreococcus strains originate from environments as diverse as the Red Sea or the coast of Brittany. We are currently extending our collection to heterotrophic picoplankton, since this compartment is probably very diversified, as revealed by recent molecular studies.

Veldhuis MJW, Kraay GW (2004) Phytoplankton in the subtropical Atlantic Ocean : towards a better assessment of biomass and composition. Deep-Sea Research Part I-Oceanographic Research Papers 51 :507-530

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A comparative study was made of different methods to determine phytoplankton composition and biomass distribution at 5 stations in the eastern Atlantic Ocean (11degrees to 35degreesN). The methods used include plant-pigment analysis and flow cytometry (size class, chlorophyll fluorescence, and phytoplankton DNA distribution). At all 5 stations a Deep Chlorophyll Maximum (DCM) at depths ranging from 80 to 130 m. As indicated by chlorophyll-a(2) content, Prochlorococcus sp. made up 25-60% of the total chlorophyll-a biomass in the euphotic zone. However, in terms of relative phytoplankton DNA and carbon content or cellular chlorophyll fluorescence, much higher values were found (45-70%) for this algal species. Prochlorococcus dominated the surface waters, but the eukaryotic phytoplankton component showed the highest biomass values at the DCM and deeper. Analysis of the accessory pigments (applying CHEMTAX) showed a clear vertical gradient at each station, but also the spatial distribution of the different taxonomic classes varied. Flow cytometric analysis of the water samples gave far less detailed information on the species/taxonomical classes present. Classification at the species level was possible only for Prochlorococcus and Synechococcus. In the case of the eukaryotes, the resolution of composition of the two main subpopulations (pico- and small eukaryotes) was enhanced after a DNA specific dye was applied. Staining the genome of the phytoplankton community revealed in most cases 5-15 separable DNA peaks, representing a similar number of different species. In general only a maximum of three DNA peaks dominated (> 500 cells per ml).
Phytoplankton carbon estimates based on DNA content matched those based on size fractionating filtration and conversion of cell size into carbon.
Phytoplankton carbon data combined with the pigment data were used to estimate the carbon to chlorophyll ratios (theta) across the light gradient for a single species (Prochlorococcus) and the eukaryotic phytoplankton community. Because of the increase in chlorophyll-a(2) content by a factor 35, the carbon to chlorophyll ratio (theta) of Prochlorococcus showed a fairly uniform pattern with a steep gradient, ranging from a maximum of 450 at the surface to 15 mug C/mug chlor. at 150 m. Furthermore, there was a south to north increase in theta by a factor of two mainly due to lower pigment values per cell of Prochlorococcus in the southern region. In contrast the theta of the collective eukaryotic phytoplankton community showed much lower values at the surface (30-80 mug C/mug chlor.), and varied with depth were far less than did theta of Prochlorococcus (only 3-7 fold variation). The reduced variability in theta of the eukaryotes can be explained by a co-variation of pigmentation and cell size. The latter can be derived from the increase in DNA content per cell with depth. Moreover, both parameters showed that the trend towards a dominance of larger species with declining growth irradiance is accompanied by a shift in community structure. This response differs greatly from that of Prochlorococcus, which tends to possess a high degree of photoacclimation enabling a single species to cover the whole euphotic zone. (C) 2004 Elsevier Ltd. All rights reserved.

Vizcaino N, Caro-Hernandez P, Cloeckaert A, Fernandez-Lago L (2004) DNA polymorphism in the omp25/omp31 family of Brucella spp. : identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes and Infection 6 :821-834

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Five genes homologous to the well-known omp25 and omp31 genes, that code for two major Brucella spp. outer membrane proteins (OMPs), have been detected in the genome of Brucella melitensis 16M and Brucella suis 1330. In this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical Brucella species reference strains and in representative strains of the recently proposed species Brucella cetaceae and Brucella pinnipediae that classify the Brucella strains isolated in the last years from marine mammals. Although these genes are quite conserved in the genus Brucella, several important differences have been found between species (i) omp31b contains a premature stop codon in B. canis and B. ovis truncating the encoded protein ; (ii) the 5’ end of omp31b is deleted in the three biovars of B. melitensis which probably prevents synthesis of Omp31b in this species ; (iii) only B. melitensis, B. suis and B. neotomae would be able to synthesize the Omp25b protein with the characteristics shared by the Omp25/Omp31 group of proteins (characteristic signal sequence and C-terminal phenylalanine) ; (iv) a DNA inversion of 1747 bp including omp25b was detected in B. cetaceae strains : (v) a DNA deletion of about 15 kb was detected in all the six B. ovis strains tested. This deletion in B. ovis includes, among other genes, omp25b and mboA, a gene that has been shown to be required for the synthesis of the O-polysaccharide chain of the Brucella spp. smooth lipopolysaccharide. Several features of the DNA region absent from B. ovis suggest that this DNA fragment is a genomic, island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. The DNA polymorphism we have found in this work within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by the Brucella species. (C) 2004 Elsevier SAS. All rights reserved.

Wang CB, Huang MQ, Tao GL, Yu GY, Han ZW, Yang ZH, Wang YJ (2004) Polypeptide from Chlamys farreri protects HaCaT cells from UVB-induced apoptosis. Chem Biol Interact 147 :119-127


A natural polypeptide from marine Chlamys farreri (a kind of scallop) (PCF), has been recently been found to be an effective photoprotective agent against ultraviolet rays B (UVB)-induced mitochondria damage in normal human fibroblasts. To investigate whether PCF has the antiapoptotic effect on human keratinocytes, in the present study, we established an apoptotic model on HaCaT cell line by means of UVB radiance of 30 mJ/cm(2) and compared the effect of different PCF treatments on UVB-radiated cells. Flow cytometry analyses showed that PCF treatment before UVB-irradiation inhibited UVB-induced apoptosis, the loss of mitochondrial membrane potential (Deltapsim) and the increase of free Ca(2+) level in HaCaT cells. In parallel with these results, UVB-irradiation enhanced activities of caspases-3, 8, 9, while this enhancement was inhibited by PCF treatment prior to irradiation. PCF added after irradiation neither reduced UVB-induced activities of the three caspases nor synergized the effect of pre-added PCF. Cellular ultrastructural features obtained from transmission electron microscopy further confirmed the antiapoptotic effect of PCF pre-treatment. It is concluded that the antiapoptotic effect of PCF is not therapeutic but prophylactic. Caspases-3, 8, 9, Deltapsim and calcium are involved in UVB-induced apoptosis, while prophylactic PCF inhibits apoptosis of UVB-irradiated HaCaT cells by blocking the caspases activities, the Deltapsim lost and the elevation of intracellular free Ca(2+) level.

Wang YC, Chaung RH, Tung LC (2004) Comparison of the cytotoxicity induced by different exposure to sodium arsenite in two fish cell lines. Aquat Toxicol 69 :67-79


Arsenic, a common environmental pollutant, is toxic to many mammalian cells. However, the arsenic-induced toxicity to aquatic animal species is unclear. This study attempted to compare the arsenic-induced cytotoxicity in various fish cells. Two fish cell lines, JF (fin cells of Therapon jarbua) and TO-2 cells (ovary cells of Tilapia), were treated with sodium arsenite in two ways to mimic acute and subacute exposure. The distinguishable alterations of cell morphology and microtubule network were observed in the cells treated by two arsenite exposure protocols. By the colony-forming assay, we demonstrated that the survival of both cell lines, treated with the high concentrations of arsenite (20-160 microM) for 2 h or with the low concentrations (0.125-10 microM) for 24 h, was decreased in a dose-dependent manner. The difference between the susceptibility of JF and TO-2 cells to arsenite was revealed by the factorial ANOVA to compare the survival rates of the arsenite-treated cells ; JF cells were more sensitive than TO-2 cells (P = 0.008 and 0.013 for the high-concentration and the low-concentration treatment, respectively). The possible mechanisms to provoke the cytotoxicity of arsenite in two cell lines were also addressed. Antioxidants, N-acetyl-cysteine and dithiothreitol, significantly prevented JF cells, but not TO-2 cells, from the arsenite-induced inhibition of survival. Additionally, apparent apoptosis of JF cells and a mitotic arrest of TO-2 cells in response to the treatment of arsenite were also demonstrated by the DNA-fragmentation analysis and the flow cytometric analysis of cell-cycle progression. The results indicate that sodium arsenite induces apoptosis in JF cells probably by causing oxidative stress and disturbs the cell cycle of TO-2 cells. These two fish cell lines can serve as the potential tools to in detail study the toxicity and the hazards of arsenic compounds to aquatic animals at molecular level in the future.

Wawrik B, Paul JH, Bronk DA, John D, Gray M (2004) High rates of ammonium recycling drive phytoplankton productivity in the offshore Mississippi River plume. Aquatic Microbial Ecology 35 :175-184

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As part of an integrated study of the regulation of carbon fixation in the offshore Mississippi River plume, we measured the rates of N-15-labeled ammonium and nitrate uptake in the surface plume waters from offshore to nearshore along the plume axis towards the Mississippi Delta. Concentrations of nitrate in the plume ranged from 0.19 to 2.5 muM with the highest concentrations primarily in the shoreward stations, while ammonium ranged from 0.17 to 0.44 muM, showing little spatial variability. Rates of ammonium uptake ranged from 16.5 to 260 nM h(-1), and showed a strong trend of increasing values from offshore towards the Mississippi Delta. In contrast, nitrate uptake rates ranged from 3.2 to 25 nM h(-1). The high rates of ammonium uptake in the presence of low ammonium concentrations and elevated nitrate was made possible by elevated rates of ammonium regeneration that exceeded ammonium uptake by 1.7 to 5.7-fold in the plume. The plume exhibited relatively low f-ratios and also contained elevated levels of Synechococcus as determined by flow cytometry and high levels of form IA (alpha-cyanobacterial) rbcL transcripts. These data suggest that a major portion of the carbon fixation observed in the offshore Mississippi River plume represents recycled production supported by high rates of ammonium regeneration.

Wen K, Ortmann AC, Suttle CA (2004) Accurate estimation of viral abundance by epifluorescence microscopy. Applied and Environmental Microbiology 70 :3862-3867

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Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% +/- 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h(-1), respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6% ; n = 6). Aldehyde fixation of samples followed by storage at 4degreesC, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h(-1) during the first hour to 0.01 h(-1) between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green 1, or SYBR Gold, and slides could be stored at -20degreesC for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and Hash frozen in liquid nitrogen upon collection and stored at -86degreesC. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.

Wetz MS, Wheeler PA (2004) Response of bacteria to simulated upwelling phytoplankton blooms. Marine Ecology-Progress Series 272 :49-57

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Until recently, studies of the fate of primary production in coastal upwelling systems have focused mainly on export through sinking of particulate organic matter (POM). In week-long deck incubations conducted during the upwelling season off Oregon, a large accumulation of carbon-rich (C:N greater than or equal to 16) dissolved organic matter (DOM) occurred following nitrate depletion by diatom blooms. The response of bacterioplankton to the DOM release in the incubations was observed using flow cytometric analysis of abundances of bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content. Relatively small increases in the abundance of HNA bacteria were observed in nitrate-replete conditions (<1.0 x 10(6) cells ml(-1)). Coincident with nitrate depletion and accumulation of the DOM, abundances and growth rates of HNA bacteria increased rapidly while little response was observed from LNA bacteria, Although growth rates and abundances of HNA cells increased markedly, a net decrease in dissolved organic carbon (DOC) was observed in only 1 incubation. Within approximately 1 d of nitrate going to depletion, HNA bacterial abundances peaked and then decreased rapidly, possibly due to flagellate grazing or viral lysing. These results indicate that on the timescale of upwelling/relaxation events, which generally last 7 to 10 d, environmental controls on bacterial populations may prevent complete degradation of phytoplankton-derived DOM, thus allowing some of this material to be exported from the system through physical processes following termination of the upwelling event.

Yang YH, Jiao NZ (2004) Dynamics of picoplankton in the Nansha Islands area of the South China Sea. Acta Oceanologica Sinica 23 :493-504

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Dynamics of major picoplankton groups, Synechococcus (Syn), Prochlorococcus (Pro), picoeukaryotes (Euk) and heterotrophic bacteria (Bact) was investigated by flow cytometry for the first time in the Nansha Islands area in the South China Sea. Averaged over the whole investigation area, depth-weighted integrated cell abundance (DWA) of Syn, Pro, Euk and Bact was 1.6 (0.4similar to5.7) x 10(3), 5.4 (0.1similar to7.3) x 10(4), 0.7 (0.2similar to2.2) x 10(3), and 2.3 (1.4similar to3.2) x 10(5) cells/mL respectively. Picoautotrophic cell abundance was low in the northwest part of the Nansha Islands where surface water temperature was low and the upper mixed layer was shallow. Concurrently, a surface maximum vertical distribution pattern was observed in this area. While in the southeast and east zones where temperatures were relatively higher and nitraclines were deeper, picoplankton is abundant and a subsurface maximum around 50similar to75 in is observed. Coupling of horizontal and vertical distribution patterns of picoplankton abundance and hydrological status was found, suggesting a strong influence of currents and water column structure on picoplankton distribution in the investigation area. Contrary to that in the shelf water in the East China Sea, the relationship between Pro and Bact in the Nansha Islands area in the South China Sea was not significantly negative but weakly positive. Moreover, a similar distribution pattern of Syn and Pro was observed. Possible reasons for these differences in the two marine regimes were discussed.

Zubkov MV, Allen JI, Fuchs BM (2004) Coexistence of dominant groups in marine bacterioplankton community - a combination of experimental and modelling approaches. Journal of the Marine Biological Association of the United Kingdom 84 :519-529

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The population dynamics in the coastal bactcrioplankton community off Plymouth (UK) was studied in samples proportionally diluted (99%, 90%, 66% and 0%) with sterile seawater, incubated in the dark for 2-4 days and monitored by flow cytometry. Nucleic acid content of cells, stained with SYBR Green I DNA specific dye, was used as an index of a genome size. Using flow sorting and fluorescence in situ hybridization (FISH) with a set of ribosomal RNA targeted oligonucleotide probes, the phylogenetic composition of dominant cytometric groups of bacterioplankton was determined to be similar during growth in the dilution series. The proportion of the low nucleic acid (LNA) group decreased and correspondingly the high nucleic acid (HNA) groups increased with dilution. The assimilation rates of free amino acids, a highly labile nutrient pool, were determined by flow sorting the dominant groups after short incubations with S-35-methionine tracer. The relative cellular amino acid assimilation by the LNA cells increased with dilution, while the activity of the HNA cells either decreased or remained unchanged. However, highly metabolically active LNA bacteria were overgrown by the HNA bacteria, presumably because the small genome size-an adaptation to living in an oligotrophic environment-did not allow the LNA group to grow sufficiently fast to compete with the HNA group under experimentally reduced grazing pressure. To examine the experimental results a numerical model of bacterioplankton population dynamics was formulated based on the hypothesis that the LNA cells consume only a labile fraction of organic nutrients (amino acids etc.), while the HNA cells feed on both the labile and more refractory sources of nutrients, and that in the absence of phytoplankton the labile source of nutrients is produced entirely by the bacterivorous flagellates. The model simulations gave credence to the hypothesized primary dependence of the LNA group on labile organic nutrients recycled within the microbial loop.

Zubkov MV, Tarran GA, Fuchs BM (2004) Depth related amino acid uptake by Prochlorococcus cyanobacteria in the Southern Atlantic tropical gyre. Fems Microbiology Ecology 50 :153-161

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Ambient concentrations and turnover rates of two amino acids, leucine and methionine, by total bacterioplankton and Prochlorococcus cyanobacteria were determined along a latitudinal transect across the Southern Atlantic gyre using a combined isotopic dilution and flow cytometric sorting technique. The ambient concentrations of methionine (0.2-0.65 nM) were about 2 times higher than the concentrations of leucine, while the turnover rates of the two amino acids were remarkably similar (0.1-0.7 nM d(-1)). The concentrations of both amino acids did not vary significantly with depth between 3 and 150 m but their turnover rates were 1.5-2 times higher in the top 3-80 m. Prochlorococcus took up amino acids in situ at high rates. Using a representative S-35-methionine precursor. about 25% of total bacterioplankton consumption of amino acids could be assigned to Prochlorococcus with low red fluorescence (Pro LRF) inhabiting the surface mixed layer down to 80 m and about 50% assigned to Prochlorococcus with high red fluorescence (Pro HRF) living below 100 m. In the same deep waters the cellular amino acid uptake of Pro LRF was less than 6% of that of the Pro HRF, indicating declining metabolic activity of the former. The mean cellular uptake rate of Pro HRF at depths below 120 m was 2.5 amol cell(-1) d(-1), 4 times higher than the rates of Pro LRF in the top 80 m. The difference could be partially explained by Pro HRF cellular biomass being twice that of Pro LRF. The biomass specific rates of Prochlorococcus were comparable or higher (particular of the Pro HRF) than that of other bacterioplankton. The reported findings could explain ecological success of mixotrophic Prochlorococcus cyanobacteria over both strictly autotrophic algae and heterotrophic bacteria in oligotrophic regions sustained by nutrient remineralisation. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.