mardi 21 avril 2009
par   G. Grégori

Allam B, Ashton-Alcox KA, Ford SE (2002) Flow cytometric comparison of haemocytes from three species of bivalve molluscs. Fish Shellfish Immunol 13 :141-158


Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.

Auffret M, Mujdzic N, Corporeau C, Moraga D (2002) Xenobiotic-induced immunomodulation in the European flat oyster, Ostrea edulis. Mar Environ Res 54 :585-589


The presence of chemical contaminants in marine coastal waters is a major subject of concern since many molecules are potentially immunotoxic, even at low concentration. During the last decade, studies in sentinel species, such as the blue mussel, Mytilus edulis, or the Pacific oyster, Crassostrea gigas, have revealed that immunosuppressive responses can be related to xenobiotic exposure, in the laboratory and in the field. In the present study, European flat oysters were experimentally exposed to heavy metals, to investigate possible alterations of their immune function. Several hematological and functional parameters of hemocytes were measured by flow cytometry, a technique allowing rapid, sensitive, cell-by-cell measurements in large cell populations. Results reveal a depression of phagocytosis and several subcellular, physiological changes in oysters exposed to cadmium alone or to cadmium and copper, suggesting an overall alteration of the phagocytic function.

Baucheron S, Grayon M, Zygmunt MS, Cloeckaert A (2002) Lipopolysaccharide heterogeneity in Brucella strains isolated from marine mammals. Research in Microbiology 153 :277-280

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Smooth lipopolysaccharides (S-LPSs) from Brucella strains isolated from seals, dolphins, porpoises, an otter and a minke whale were characterized by ELISA using monoclonal antibodies (mAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes and by Western blot after SDS-PAGE. All strains studied were A-dominant as shown by specific polyclonal sera and this was also confirmed by the mAbs. However, binding patterns in ELISA of mAbs to the specific common (C) epitopes were rather heterogeneous, and for some strains, such as those isolated from striped dolphins, binding of these mAbs was much reduced or negative as had previously been shown for Brucella suis biovar 2 strains. Western blot after SDS-PAGE showed the typical A-dominant strain banding pattern for all marine mammal Brucella isolates, but the average S-LPS size was shorter in many of these compared to reference Brucella abortus strain 544. Thus, S-LPSs of the marine mammal isolates show heterogeneity with regard to their O-PS C epitope content and their average size. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Becker A, Meister A, Wilhelm C (2002) Flow cytometric discrimination of various phycobilin-containing phytoplankton groups in a hypertrophic reservoir. Cytometry 48 :45-57

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Background : Knowledge of phytoplankton structure is important information in water quality control. Lake restoration and sanitation measures in particular must be evaluated on the organismic level to valuate biological effects and assess the risk of potentially toxic Cyanobacteria blooms. We used and comparatively tested three independent methods for phytoplankton analysis in a hypertrophic reservoir under restoration.
Methods : Nine unialgal cultures and outdoor samples were examined by high-performance liquid chromatography pigment analysis, microscopical cell counting, and flow cytometric (FCM) light scatter and fluorescence analysis to measure the percentage contribution of the major algal groups to chlorophyll a and biovolume, The FCM instrument settings and identification criteria were developed using a single excitation wavelength at 514 nm to differentiate nine algal species representing the major groups of algae. Fluorescence was detected at 585, 620, 650, and 680 nm.
Results : The results show that FCM is the only method for determining changes in the phytoplankton composition on both a chlorophyll a and biovolume basis.
Conclusions : Each of the three methods has specific advantages and disadvantages, and should be chosen depending on the experimental problem. FCM sorting allows the combination of all three and offers further new perspectives. (C) 2002 Wiley-Liss, Inc.

Bihari N, Hamer B, Jaksic Z, Fafandel M, Micic M, Batel R (2002) Application of alkaline elution, Fast Micromethod and flow cytometry in detection of marine contamination. Cell Mol Biol (Noisy-le-grand) 48 :373-377


DNA damage is an inescapable aspect of life in the biosphere. The presented investigations were an attempt to examine the response of a DNA damage as a biomarker of environmental quality in the mussels Mytilus galloprovincialis sampled at differently contaminated areas of Istrian coast, Northern Adriatic. The investigations were performed in order to get information about the genotoxic risk for marine organisms exposed to mixed environmental pollution, as well as the information about the presence of unknown mixture of genotoxic contaminants in the marine environment. Types of DNA damage detected are alkali-labile sites and single-strand breaks measured by Fast Micromethod, interstrand cross-links and DNA protein cross-links by alkaline filter elution and cell cycle disturbation by flow cytometry. The applicability of all three methods for marine quality control is discussed.

Boelen P, Post AF, Veldhuis MJ, Buma AG (2002) Diel patterns of UVBR-induced DNA damage in picoplankton size fractions from the Gulf of Aqaba, Red Sea. Microb Ecol 44 :164-174


This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation : 280 to 315 nm) on bacterio- and phytoplankton (<10 microm) from the Gulf of Aqaba, Red Sea. Incident biologically effective doses (BEDs) and attenuation of biologically effective radiation in the water column were measured using a DNA biodosimeter. UVBR-induced DNA damage was measured as cyclobutane pyrimidine dimers (CPDs), using an antibody directed to CPDs followed by chemiluminescent detection. Depth profiles of DNA damage were determined in two plankton size fractions (0.2 to 0.8 microm and 0.8 to 10 microm) collected down to 50 m depth. Furthermore, accumulation and removal of CPDs were monitored in surface plankton samples during several daily cycles. Small plankton (plankton <10 microm) composition was determined by flow cytometry. The plankton community in the Gulf of Aqaba was dominated by nonphototrophic bacteria and the free-living prochlorophyte Prochlorococcus spp. (<0.8 microm). In general, no DNA damage could be detected in dosimeter DNA below 15 m. In contrast, DNA damage (up to 124 CPD Mnucl-1) could be detected in all bacterio- and phytoplankton samples. DNA damage accumulated throughout the day, indicating that plankton in the Gulf of Aqaba undergo UVBR stress via CPD induction. Although the numbers of CPDs decreased during darkness, both size fractions showed some residual DNA damage at the end of the night. This suggests that dark repair processes did not remove all CPDs, or that part of the plankton community was incapable of repair at all. CPD levels in the two size fractions showed no significant differences in situ. During full solar radiation exposures (samples incubated in bags), more CPDs were detected in the smaller (0.2 to 0.8 microm) size fraction as compared to the larger (0.8 to 10 microm) size fraction. In these experiments, initial plankton composition was significantly different from the field samples. This implies that a shift in the population structure or irradiance conditions can lead to a significant change in UVBR sensitivity. In conclusion, the results show that the picoplankton-dominated phyto- and bacterioplankton communities in the clear surface waters from the Gulf of Aqaba undergo UVBR stress. Repair pathways are not sufficient to eliminate damage during or after UVBR exposure hours, suggesting photomortality as a potential loss parameter of the plankton community.

Boelen P, Post AF, Veldhuis MJW, Buma AGJ (2002) Diel patterns of UVBR-induced DNA damage in picoplankton size fractions from the Gulf of Aqaba, Red Sea. Microbial Ecology 44 :164-174

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This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation : 280 to 315 nm) on bacterio- and phytoplankton (<10 μm) from the Gulf of Aqaba, Red Sea. Incident biologically effective doses (BEDs) and attenuation of biologically effective radiation in the water column were measured using a DNA biodosimeter. UVBR-induced DNA damage was measured as cyclobutane pyrimidine dimers (CPDs), using an antibody directed to CPDs followed by chemiluminescent detection. Depth profiles of DNA damage were determined in two plankton size fractions (0.2 to 0.8 μm and 0.8 to 10 μm) collected down to 50 m depth. Furthermore) accumulation and removal of CPDs were monitored in surface plankton samples during several daily cycles. Small plankton (plankton < 10 mum) composition was determined by flow cytometry. The plankton community in the Gulf of Aqaba was dominated by nonphototrophic bacteria and the free-living prochlorophyte Prochlorococcus spp. (<0.8 μm). In general, no DNA damage could be detected in dosimeter DNA below 15 m. In contrast, DNA damage (up to 124 CPD Mnucl(-1)) could be detected in all bacterio- and phytoplankton samples. DNA damage accumulated throughout the day, indicating that plankton in the Gulf of Aqaba undergo UVBR stress via CPD induction. Although the numbers of CPDs decreased during darkness, both size fractions showed some residual DNA damage at the end of the night. This suggests that dark repair processes did not remove all CPDs, or that part of the plankton community was incapable of repair at all. CPD levels in the two size fractions showed no significant differences in situ. During full solar radiation exposures (samples incubated in bags), more CPDs were detected in the smaller (0.2 to 0.8 μm) size fraction as compared to the larger (0.8 to 10 μm) size fraction. In these experiments, initial plankton composition was significantly different from the field samples. This implies that a shift in the population structure or irradiance conditions can lead to a significant change in UVBR sensitivity. In conclusion, the results show that the picoplankton-dominated phyto- and bacterioplankton communities in the clear surface waters from the Gulf of Aqaba undergo UVBR stress. Repair pathways are not sufficient to eliminate damage during or after UVBR exposure hours, suggesting photomortality as a potential loss parameter of the plankton community.

Bolter M, Bloem J, Meiners K, Moller R (2002) Enumeration and biovolume determination of microbial cells - a methodological review and recommendations for applications in ecological research. Biology and Fertility of Soils 36 :249-259

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A review of direct counts of micro-organisms is presented, which describes the evaluation of different techniques and applications of microscopic methods for enumeration, biovolume and biomass measurement. New staining techniques and their applications are discussed as well as methods to distinguish individual cell properties. Attention is paid to data analysis and error propagation, which is widely neglected during quantitative studies in microbial ecology. Comparisons to other methods are made in order to evaluate methods of direct microscopy with respect to their great importance for environmental descriptions and analyses.

Buesing N, Gessner MO (2002) Comparison of detachment procedures for direct counts of bacteria associated with sediment particles, plant litter and epiphytic biofilms. Aquatic Microbial Ecology 27 :29-36

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Efficient detachment of bacterial cells is crucial for assessing bacterial abundance, biomass and community composition in natural and technical systems (e.g. sewage plants) by a wide range of analytical methods. There is no agreement on which procedure gives the best results with which type of substratum. We tested the effect of 4 detachment instruments on the release of bacteria associated with leaf litter, sediment and epiphytic biofilms from a natural aquatic system. Treatment with most instruments increased bacterial counts and biovolumes significantly compared to simple vortexing (by 2.7 to 7x). However, both the numbers and biovolume of released bacterial cells varied significantly between detachment devices. With leaf litter and epiphytic biofilms, an ultrasonic probe treatment released 10 and 4 times more bacterial cells than the most inefficient instrument. A stomacher-type blender gave the best results for sediment samples, releasing 3 times higher numbers of bacteria than the least efficient instrument. Neither the detachment instrument nor the treatment time affected the composition of bacterial morphotypes. These results indicate that the choice of the appropriate detachment device depends critically on the type of substratum examined, when the absolute abundance or biomass of bacteria is to be determined. In qualitative analyses of bacterial community structure the chosen device appears to be less important.

Cadee GC, Hegeman J (2002) Phytoplankton in the Marsdiep at the end of the 20th century ; 30 years monitoring biomass, primary production, and Phaeocystis blooms. Journal of Sea Research 48 :97-110

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Regular phytoplankton research in the Marsdiep started in the early 1970s as a curiosity-driven project fitting in the International Biological Programme (IBP). Interest was primarily in seasonal variation in species composition, biomass and primary production. Initially our monitoring was not intended to extend beyond the IBP programme, which ended in 1971. However, the example and support of Jan Beukema and the interesting links with his work on secondary production in the Wadden Sea were decisive. in its continuation. As a result, we now have a time series of well over 30 years. We had the good luck to start in a period of relatively low phytoplankton values, whereas in the late 1970s biomass and primary production doubled in a short period. Duration of the Phaeocystis blooms, which regularly occur in spring and early summer in Dutch coastal waters, also increased. Light limitation plays a dominant role in turbid coastal waters. Our Secchi disc data up to 1985 did not indicate changes in turbidity, and therefore increases were seen as a eutroohication phenomenon. What would then be more natural than to expect phytoplankton to decrease again with the lowering of phosphate values in the 1980s and 1990s as a result of the successful cleaning of the Rhine ? The data up to 1992 did not show any effect on phytoplankton of this deeutrophication. Annual primary production during the period 1992-2000 decreased from a peak value of >400 gC m(-2) in 1994 to ca. 250 now. Over the entire period 1974-2000, a slight increase in turbidity was observed. Phosphate concentrations have dropped a little further since 1992, but as could be expected, dissolved nitrogen (nitrate, nitrite and ammonia) and reactive silica did not show changes since the mid 1970s ; inputs from freshwater and the atmosphere have not decreased. In accordance with the decrease of primary production, also chlorophyll-a and Phaeocystis cell numbers have dropped since 1994. These first signs of a decrease in phytoplankton parameters probably related to de-eutrophication make continuation of our Marsdiep monitoring highly interesting. No lengthening of the growing season of phytoplankton was observed comparable to that observed in terrestrial vegetation and related to global warming, but Phaeocystis growth seems to start earlier now. Our phytoplankton research covers only a small part of the period of man-induced changes of the Wadden Sea ecosystem, which started already in the Middle Ages. Certainly the recent overfishing of filter feeders such as cockles and mussels has affected phytoplankton, but also the cultivation of mussels and introduction of exotics such as Crassostrea gigas and Ensis directus must have influenced phytoplankton. The Marsdiep phytoplankton time series has proved to be useful in the continued ecosystem research in the western Wadden Sea. Continuation of this time series will also be helpful to study natural versus human-induced variations in this area. (C) 2002 Elsevier Science B.V All rights reserved.

Casamayor EO, Pedros-Alio C, Muyzer G, Amann R (2002) Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations. Appl Environ Microbiol 68 :1706-1714


Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques : epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.

Christaki U, Courties C, Karayanni H, Giannakourou A, Maravelias C, Kormas KA, Lebaron P (2002) Dynamic characteristics of Prochlorococcus and Synechococcus consumption by bacterivorous nanoflagellates. Microbial Ecology 43 :341-352

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We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 mum in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) 3 to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.

Coteur G, DeBecker G, Warnau M, Jangoux M, Dubois P (2002) Differentiation of immune cells challenged by bacteria in the common European starfish, Asterias rubens (Echinodermata). Eur J Cell Biol 81 :413-418


Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.

del Giorgio PA, Bouvier TC (2002) Linking the physiologic and phylogenetic successions in free-living bacterial communities along an estuarine salinity gradient. Limnology and Oceanography 47 :471-486

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In this paper we assess whether the bacterial compositional succession that occurs along an estuarine salinity gradient is accompanied by changes in both community and single-cell metabolic activity in the free-living bacterioplankton. In addition, we explore whether up- and downstream estuarine communities, which are dominated by different phylogenetic groups, show distinct patterns in single-cell activity and characteristics. We have characterized the physiological succession of the bacterioplankton along the salinity gradient in the Choptank River Estuary (Maryland), using a combination of indices of single-cell activity, which include respiratory activity, membrane polarization and integrity, and DNA and RNA contents, combined with flow cytometry. We have also measured bacterioplankton community production, respiration, and growth efficiency along the estuary. Our data suggest that the sharp phylogenetic succession that occurs within the fresh to saltwater transition region is accompanied by profound metabolic changes both at the single-cell and community levels and that the phylogenetic succession occurs together with measurable physiological stress. The different indices of single-cell characteristics that we have used converge to suggest that within the fresh to saltwater transition zone, there is generalized decline in growth and possibly a loss of activity and even significant cell mortality. At the community level, these changes in single-cell physiology at the site of the phylogenetic succession appear to translate into a generalized decline in bacterial growth efficiency with a decrease in bacterial growth and production but an actual increase in bacterial carbon consumption. Our data also suggest that different phylogenetic groups may have intrinsically different levels of single-cell activity or at least respond differently to the activity assays that we currently use.

DuRand MD, Green RE, Sosik HM, Olson RJ (2002) Diel variations in optical properties of Micromonas pusilla (Prasinophyceae). Journal of Phycology 38 :1132-1142

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Micromonas pusilla (Butcher) Manton et Parke, a marine prasinophyte, was used to investigate how cell growth and division affect optical properties of phytoplankton over the light:dark cycle. Measurements were made of cell size and concentration, attenuation and absorption coefficients, flow cytometric forward and side light scattering and chl fluorescence, and chl and carbon content. The refractive index was derived from observations and Mie scattering theory. Diel variations occurred, with cells increasing in size, light scattering, and carbon content during daytime photosynthesis and decreasing during nighttime division. Cells averaged 1.6 mum in diameter and exhibited phased division, with 1.3 divisions per day. Scattering changes resulted primarily from changes in cell size and not refractive index ; absorption changes were consistent with a negligible package effect. Measurements over the diel cycle suggest that in M. pusilla carbon-specific attenuation varies with cell size, and this relationship appears to extend to other phytoplankton species. Because M. pusilla is one of the smallest eukaryotic phytoplankton and belongs to a common marine genus, these results will be useful for interpreting in situ light scattering variation. The relationship between forward light scattering (FLS) and volume over the diel cycle for M. pusilla was similar to that determined for a variety of phytoplankton species over a large size range. We propose a method to estimate cellular carbon content directly from FLS, which will improve our estimates of the contribution of different phytoplankton groups to productivity and total carbon content in the oceans.

Ferris MM, McCabe MO, Doan LG, Rowlen KL (2002) Rapid enumeration of respiratory viruses. Analytical Chemistry 74 :1849-1856

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Virus detection and enumeration has become increasingly important in fields ranging from medicine and biotechnology to environmental science. Although there are a wide variety of techniques that can be used to count viruses, there is demand for a rapid and more accurate means for virus enumeration. In this work, the performance of a flow cytometer that was designed and custom-built specifically for rapid detection of single viruses was evaluated. The instrument, designated a single nanometric particle enumerator (SNaPE), was characterized and calibrated using fluorescent polystyrene nanospheres. The reliability of the instrument with respect to virus enumeration was demonstrated for three medically relevant viruses, adenovirus-5, respiratory syncytial virus, and influenza A, treated with a fluorescent nucleotide stain. In each case, the SNaPE yielded a virus particle concentration consistent with, but slightly lower than, transmission electron microscopy (TEM) results, as expected. In addition, oil the basis of calibration of signal intensity, the average peak height for a given virus was correlated with genome size, as expected. In contrast to time-consuming analyses such as TEM and plaque titers, SNaPE analysis of pure virus samples (including sample handling, data collection, and data processing) can be completed within 1 h.

Ferris MM, Rowlen KL (2002) Detection and enumeration of single nanometric particles : A confocal optical design for fluorescence flow cytometry. Review of Scientific Instruments 73 :2404-2410

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Nanometer-sized particles are increasingly important in fields ranging from technology development to environmental analysis. Traditionally, quantification of biological nanometric particles, such as viruses, has been challenging. Recent advances in optics and optical design have made single molecule detection possible in flowing systems. However, many commercially available flow cytometers are not optimized for routine analysis of ultrasmall biological particles. In this work, a confocal optical arrangement was incorporated into the design of a relatively simple flow cytometer optimized for rapid enumeration of fluorescent nanometric particles. The instrument was designated the "single nanometric particle enumerator" (SNaPE). Instrument parameters, such as data acquisition rates, flow rates, and minimum sampling time were examined and optimized for the SNaPE. The measured detection efficiency was similar to8%, which is consistent with the confocal geometry and equivalent to the detection efficiency reported in many single molecule studies. Signal calibration was achieved using fluorescent polystyrene spheres ranging from 26 nm to 10 mum in diameter. The SNaPE exhibited a limit of detection of similar to180 fluorescein isothiocyanate equivalents and was capable of particle enumeration over the tested concentration range of 10(6)-10(9) particles/ml. (C) 2002 American Institute of Physics.

Ferris MM, Stoffel CL, Maurer TT, Rowlen KL (2002) Quantitative intercomparison of transmission electron microscopy, flow cytometry, and epifluorescence microscopy for nanometric particle analysis. Analytical Biochemistry 304 :249-256

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Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerate. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM) ; each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating less than or equal to5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM. (C) 2002 Elsevier Science (USA).

Fitch AJ, Kolesik P, Pile AJ, Goodman AE (2002) Plasmid maintenance and localisation of Vibrio sp S141(p519ngfp) cells within monoculture and mixed-species biofilms. Biofouling 18 :275-283

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This study examined cell localisation and plasmid maintenance of Vibrio sp. S141(p519ngfp) cells which grew in, and detached from, monoculture and mixed-species marine biofilms under continuous flow conditions. Over the 48 h time course of the experiments, the broad host range IncQ RSF1010 derivative plasmid, p519ngfp, was maintained in S141(p519ngfp) cells detaching from the biofilms irrespective of selection for the plasmid, the presence of another bacterial species, or the order of substratum colonisation. S141(p519ngfp) cell localisation within mixed-species biofilms was affected by the order, and length of time, of colonisation. When S141(p519ngfp) was the initial coloniser of the biofilm, the localisation of the majority of the plasmid-bearing cells near the substratum surface was not affected. When S141(p519ngfp) cells colonised a pre-existing Psychrobacter sp. SW5HR biofilm, or the two species simultaneously colonised the substratum, non plasmid-bearing cells initially dominated the substratum surface ; as the time of S141(p519ngfp) colonisation increased, the plasmid-bearing cells appeared to displace the SW5HR cells from the surface. Since the p519ngfp plasmid was stably maintained in the Vibrio sp. S141 host forming biofilms over a 48 h period, GFP-producing S141 cells were able to be localised in mixed-species biofilms and were found to dominate the substratum surface by 48 h.

Fournier M, Pellerin J, Lebeuf M, Brousseau P, Morin Y, Cyr D (2002) Effects of exposure of Mya arenaria and Mactromeris polynyma to contaminated marine sediments on phagocytic activity of hemocytes. Aquat Toxicol 59 :83-92


Two species of bivalves, Mya arenaria and Mactromeris polynima, were exposed to contaminated marine sediments from Baie des Anglais, Quebec, for a period of 10 and 12 weeks, respectively, in order to determine if there was an effect on the phagocytic activity of hemocytes from each species. These sediments contain elevated levels of both PAHs and PCBs. Uncontaminated beach sand was used as control sediments. After a period of 4 weeks, each species of bivalves were sampled and hemocyte phagocytic activity was monitored by flow cytometry. While phagocytosis by hemocytes from M. polytiyma was significantly suppressed, those from M. arenaria were not different from beach sand-exposed controls. At the end of the exposure period, the phagocytic activity of hemocytes from both species was suppressed. Physiological parameters such as mantle proteins or malondialdehyde levels, total protein and total glycogen levels in the digestive gland were not affected by exposure to contaminated sediments. Moreover, the suppression of phagocytosis was well correlated with the transfer of contaminants from the sediments to the bivalves and their subsequent bioaccumulation, as demonstrated by the PCB body burden. These results support the use of bivalves as good sentinel species to survey sediment contamination and the usefulness of hemocyte phagocytic activity as a sensitive biomarker of exposure to organic contaminants.

Franklin NM, Stauber JL, Apte SC, Lim RP (2002) Effect of initial cell density on the bioavailability and toxicity of copper in microalgal bioassays. Environ Toxicol Chem 21 :742-751


Algal toxicity tests based on growth inhibition over 72 h have been extensively used to assess the toxicity of contaminants in natural waters. However, these laboratory tests use high cell densities compared to those found in aquatic systems in order to obtain a measurable algal response. The high cell densities and test duration can result in changes in chemical speciation, bioavailability, and toxicity of contaminants throughout the test. With the recent application of flow cytometry to ecotoxicology, it is now possible to use lower initial cell densities to minimize chemical speciation changes. The speciation and toxicity of copper in static bioassays with the tropical freshwater alga Chlorella sp. and the temperate species Selenastrum capricornutum (Pseudokirchneriella subcapitata) were investigated at a range of initial cell densities (10(2)-10(5) cells/ml). Copper toxicity decreased with increasing initial cell density. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h median effective concentration [EC50]) increased from 4.6 to 16 microg/L for Chlorella sp. and from 6.6 to 17 microg/L for S. capricornutum as the initial cell density increased from 10(2) to 10(5) cells/ml. Measurements of anodic stripping voltammetry-labile, extracellular, and intracellular copper confirmed that at higher initial cell densities, less copper was bound to the cells, resulting in less copper uptake and lower toxicity. Chemical measurements indicated that reduced copper toxicity was due primarily to depletion of dissolved copper in solution, with solution speciation changes due to algal exudates and pH playing a minor role. These findings suggest that standard static laboratory bioassays using 10(4) to 10(5) algal cells/ml may seriously underestimate metal toxicity in natural waters.

Gasol JM, Comerma M, Garcia JC, Armengol J, Casamayor EO, Kojecka P, Simek K (2002) A transplant experiment to identify the factors controlling bacterial abundance, activity, production, and community composition in a eutrophic canyon-shaped reservoir. Limnology and Oceanography 47 :62-77

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We performed a transplant experiment in eutrophic Sau reservoir to assess the factors that control bacterial abundance. activity, growth rate, and community composition. Samples from the lacustrine and the riverine ends of the reservoir were incubated in dialysis bags placed in situ and transplanted to the other side of the reservoir and also incubated after 1 mum filtration to measure predator effects. The bags were sampled at 12-h intervals to estimate bacterial abundance, whole community activity, activity structure (by flow cytometry), and phylogenetic composition (by in situ hybridization with group-specific phylogenetic probe.). Bacterial production was always regulated by nutrient supply, but abundance and activity were differently regulated at both sites. The riverine bacteria were limited by predator activity, whereas the lacustrine were regulated by a combination of predation and nutrient supply. Therefore, even in the same environment, different modes of control can act simultaneously. Bacterial activity structure was also regulated in the same way. Abundance of highDNA bacteria and cells hybridizing with the universal EUB338 probe were well correlated. In the lacustrine sample, bacterial community structure did not change significantly, whereas in the riverine sample, alpha- and gamma-Proteobacteria reduced their growth when transplanted, whereas beta-Proteobacteria were stimulated by the presence of predators. Members of the Cytophaga/Flavobacterium phylum grew only when incubated in situ in the absence of predators. This different behavior in the different bacterial groups resulted in strong changes in bacterial assemblage composition, evident already after 24 h. The experiment demonstrates that, together with the effect of predators, nutrient supply affects bacterial community properties and that a complex regulation involving both types of control can occur in a single heterogeneous planktonic system.

Gregori G, Denis M, Lefevre D, Beker B (2002) A flow cytometric approach to assess phytoplankton respiration. Methods Cell Sci 24 :99-106


Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta.

Groben R, Medlin LK (2002) Meeting report : EU workshop "Analysis of Single Cells in the Marine Phytoplankton" (ASCMAP), Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany, April 15-21, 2002. Protist 153 :193-195


Holtzendorff J, Marie D, Post AF, Partensky F, Rivlin A, Hess WR (2002) Synchronized expression of ftsZ in natural Prochlorococcus populations of the Red Sea. Environ Microbiol 4 :644-653


The expression of ftsZ, encoding the initiating protein of the prokaryotic cell division was analysed in natural Prochlorococcus populations in the Gulf of Aqaba, northern Red Sea. During the seasonal Prochlorococcus bloom in September 2000, picoplankton was collected from the deep chlorophyll maximum (DCM) at 2-4 h intervals over 3 consecutive days. Flow cytometric measurements as well as DNA sequence analyses showed that Prochlorococcus was the dominant photosynthetic organism. Cell densities peaked as high as 1.4 x 10(5) cells ml(-1). This DCM population mainly consisted of brightly red fluorescing Prochlorococcus cells, corresponding to low light-adapted ’ecotypes’ (sensu Moore et al., 1998, Nature 393 : 464-467). Prochlorococcus populations grew in a highly synchronized fashion with DNA replication in the afternoon and cell division during the night. The ftsZ mRNA level reached maximum values within the replication phase between 14.00 and 16.00 hours, and minimum values between 02.00 and 06.00 hours. Thus, the transcriptional regulation of ftsZ could be a major factor triggering the synchronized cell division of Prochlorococcus populations. This is the first application of quantitative reverse transcriptase-coupled real-time polymerase chain reaction (PCR) to natural populations of an environmentally relevant marine organism.

Jacquet S, Havskum H, Thingstad TF, Vaulot D (2002) Effects of inorganic and organic nutrient addition on a coastal microbial community (Isefjord, Denmark). Marine Ecology-Progress Series 228 :3-14

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Using flow cytometry (FCM), microbial populations were followed in a mesoscosm experiment manipulated with daily additions of mineral nutrients (as phosphates and nitrates in Red-field equilibrium), of degradable organic carbon (as glucose-C), or with the 2 treatments combined. Intensive sampling was performed in order to assess the short time-scale variability of the microbial community. Five autotrophic groups (including Synechococcus spp, and cryptophytes), 2 groups of heterotrophic bacteria, and 2 groups of viruses could be discriminated by FCM. The control enclosure (no addition) revealed that heterotrophic bacteria were carbon-limited. Synechococcus spp. abundance increased in the control, presumably because they experienced little competition from heterotrophic bacteria (which were C-limited) and from larger phytoplankton (which were not as efficient in nutrient uptake at low nutrient concentration and could not, therefore, sustain high growth rates). When N and P were added, however, larger-celled autotrophic populations were favoured. When glucose was added, alone or together with inorganic elements, the abundance of Synechococcus spp. and small eukaryotes was reduced, suggesting that, when released from C-limitation, heterotrophic bacteria are the best competitors for mineral nutrients. The addition of both inorganic and organic nutrients also enhanced cryptophytes in contrast to all other autotrophs, probably because of their heterotrophic capacity. Our results agree with recent evidence suggesting that heterotrophic bacteria are limited by both carbon and mineral nutrients, and demonstrate how this has important consequences for the success of their trophic neighbours in the microbial food web.

Jacquet S, Heldal M, Iglesias-Rodriguez D, Larsen A, Wilson W, Bratbak G (2002) Flow cytometric analysis of an Emiliana huxleyi bloom terminated by viral infection. Aquatic Microbial Ecology 27 :111-124

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During a field mesocosm experiment conducted in coastal waters off western Norway, 11 m(3) enclosures were filled with unfiltered seawater and enriched daily with different nitrate and phosphate concentrations in order to induce a bloom of the coccolithophorid Emiliana huxleyi under different nutrient regimes. Flow cytometry (FCM) analysis was performed 5 times d(-1) in order to follow the initiation, development and termination of the bloom as well as the production of large virus-like particles (LVLPs) identified as E, huxleyi viruses (EhV). EhV production was observed first in the enclosure where N was in excess, and P limitation induced a lower burst size compared to nitrate-replete enclosures. These observations suggest a critical role for both P and N in E. huxleyi-EhV interactions. Concomitant to EhV production, a shift was observed between the original population (coccolith-bearing cells) towards a population characterized by the same chlorophyll a (chl a) fluorescence but with lower right angle light scatter values. This population is likely to correspond to either senescent cells losing their coccoliths or cells characterized by a lower production of coccoliths possibly due to viral infection, At the end of experiment, a significant proportion of E. huxleyi had survived after the end of the bloom. This suggests either the presence of a resistant form of the coccolithophorid or a change in the dominance of different host and/or viral strains during the bloom. A periodical pattern in virus production was recorded with virus number decreasing during the second part of the day suggesting that virus production may be synchronized to the daily light cycle. Our results provide new insights towards the understanding of the relationship between a key marine species and its specific virus.

Jacquet S, Prieur L, Avois-Jacquet C, Lennon JF, Vaulot D (2002) Short-timescale variability of picophytoplankton abundance and cellular parameters in surface waters of the Alboran Sea (western Mediterranean). Journal of Plankton Research 24 :635-651

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The Alboran Sea (western Mediterranean) is characterized by a well-defined hydrological structure, the Almeria (Spain)-Oran (Algeria) geostrophic front. During the Almofront-2 cruise (November 22, 1997 to January 18, 1998), high frequency sampling (Deltat = 0.5 h) of autotrophic picoplankton in surface waters was performed for 17 days (December 24 to January 9) using an automatic sampler. Cell abundance and several cellular parameters were measured by flow cytometry (FCM) : forward and right-angle light scatters (FALS, RALS), phycoerythrin (PE) and chlorophyll (Chl) fluorescence. Analysis of abundance of Prochlorococcus, Synechococcus and picoeukaryotes allowed the distinction of two major types of systems : mesotrophic conditions (with waters characterized by detectable nutrients) dominated by picoeukaryotes and more oligotrophic areas (with low to undetectable nutrient levels) dominated by Prochlorococcus and, to a lower extent, Synechococcus. Most of the cellular parameters exhibited a clear diel periodicity, suggesting that cell growth and division processes were tightly coupled to the daily light cycle despite strong gradients of temperature and salinity. Cell growth for both Synechococcus and picoeukaryotes began at dawn and stopped at dusk. In contrast, the increase of Prochlorococcus light scattering only began in late afternoon, a very unusual behaviour that was apparently associated with strong quenching of Prochlorococcus Chl fluorescence during the day. Our data also suggested Chl quenching in Synechococcus but not in picoeukaryotes. Fourier analysis established unambiguously the 24 h diel periodicity for all cellular parameters as well as for cryptophyte abundance, but not for the abundance of other phytoplankters. The burst of division for picophytoplanktonic cells, inferred from the timing of the decrease of light scatter, occurred at dusk for both Synechococcus and picoeukaryotes, and later at night for Prochlorococcus. The ratio between maximum and minimum scatter (RALS(max/min)) was useful in assessing the physiological state of the different picoplankters : high values suggested that populations probably had high division rates, especially picoeukaryotes, suggesting a quasi-optimal physiological activity of the cells in all water types encountered during this cruise despite strong hydrological gradients. This study reveals that the growth rate of the populations may be very little in these ecosystems.

Jiao NZ, Yang YH, Koshikawa H, Watanabe M (2002) Influence of hydrographic conditions on picoplankton distribution in the East China Sea. Aquatic Microbial Ecology 30 :37-48

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An investigation was made on picoplankton distributions in relation to physical and chemical conditions in the East China Sea, a marginal sea of the Northwest Pacific, in July 1998. Synechococcus, pico-eukaryotes and heterotrophic bacteria were ubiquitous, with average abundance at the order of 10(4), 10(2) and 10(5) cells ml(-1), respectively. Prochlorococcus was present at most locations beyond the 50 m isobath at around 10(4) Cells ml(-1). Responses of these picoplankters to the hydrographic conditions were evident in both vertical and horizontal distributions. Prochlorococcus were basically associated with oceanic warm currents, and sudden changes in cell abundance often occurred within a relatively short distance of the currents. In the surface mixed layer, Prochlorococcus were usually present only when temperature was >26degreesC, salinity >30 psu, total inorganic nitrogen < 3 muM and phosphate <0.4 muM in the study period. Vertically, however, Prochlorococcus were distributed down where temperature was as low as 16degreesC and nutrient levels were also higher. No pronounced subsurface peaks in Prochlorococcus abundance were recorded in the oceanic warm currents although Prochlorococcus outnumbered Synechococcus by at least an order of magnitude. Synechococcus were most abundant in the coastal area associated with high nutrient levels. Pico-eukaryotes usually developed very well in the front areas on the continental shelf. Along offshore directions, pico-eukaryotes often centered farther from the shore and deeper in the water column than did Synechococcus. Heterotrophic bacteria showed the least variation in abundance among the 4 picoplankters, but still decreased distinctly in offshore directions, following a similar trend in the total biomass of pico-eukaryotes and Synechococcus. In the Yangtze River plume area, light availability was also important in regulating picoplankton distribution patterns. The relationship between Prochlorococcus and bacteria biomass was negative along gradients in the marginal sea.

Juhl AR, Latz MI (2002) Mechanisms of fluid shear-induced inhibition of population growth in a red-tide dinoflagellate. Journal of Phycology 38 :683-694

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Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red-tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5-mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette-flow chambers (0.004-0.019 N.m(-2) shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA.cell(-1). The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential-phase cultures exposed to the lowest experimental shear stress in Couette-flow chambers. However, mortality occurred when a late exponential-phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near-surface waters. Shear-induced mortality is not expected unless shear-stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.

Kawai M, Matsutera E, Kanda H, Yamaguchi N, Tani K, Nasu M (2002) 16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis. Appl Environ Microbiol 68 :699-704


The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.

Landry MR, Kirchman DL (2002) Microbial community structure and variability in the tropical Pacific. Deep-Sea Research Part Ii-Topical Studies in Oceanography 49 :2669-2693

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The spatially extensive tropical Pacific includes regions that are limited by macronutrients or iron, and is thus broadly representative of open-ocean systems in which microbial communities predominate. Despite strong physical forcing due to the El Nino-Southern Oscillation cycle and the local effects of tropical instability waves, microbial abundances from a variety of JGOFS and related studies show similar, modest levels of variability in the high-nutrient, low-chlorophyll (HNLC) equatorial upwelling region, the oligotrophic, western Pacific Warm Pool, and the North Pacific central gyre. Mean 0-50m abundances of some of the groups distinguished by flow cytometry are significantly enhanced in the HNLC region, including heterotrophic bacteria (HBACT ; 720,000 versus 440,000 cells ml(-1)), Synechococcus spp. (SYN ; 9800 versus 2000 cells ml(-1)) and pico-eukaryotic algae (PEUK ; 6300 versus 800 cells ml(-1)). However, Prochlorococcus spp. (PRO) are slightly more abundant in the low-nitrate regions (180,000 versus 150,000 cells ml(-1)). The higher HNLC concentrations of SYN and PEUK are part of a broader expansion of the phytoplankton community over the relatively constant PRO base when the limiting nutrient (iron) pool is increased. Elevated biomass and production of phytoplankton and the greater availability of DOC presumably explain the higher HNLC abundances of HBACT. The mean biomass (+/-standard deviation) of bacterial populations for cross-equatorial transects (14.1+/-2.8 mug Cl-1) is similar to that in the subtropics (11.6+/-2.7 mug CI-1), with cruise variations falling generally within a 2-fold range. Heterotrophs comprise a significantly higher mean percentage of total prokaryote biomass (59+/-9%) in the HNLC region than in the low-nutrient subtropics (42+/-6%). The biomass production of photosynthetic bacteria (PRO and SYN) in the central equatorial Pacific is conservatively twice that of HBACT, but total carbon flux through bacteria (44-75% of phytoplankton C-14-production) is dominated by the high respiration, hence carbon demand, of heterotrophs. Given, the very different growth limiting factors (Fe, N, P, and organic carbon) among the various subregions and microbial groups in the tropical Pacific, it seems unlikely that direct controls on growth rate are sufficiently precise to account for the relatively low microbial variability observed. Among factors affecting loss rates, the regulatory role of viral lysis remains largely unexplored, as in most open-ocean systems. However, there is relatively good evidence, including the grazing response to the IronEx II perturbation and multi-level cascade influences, that protistan grazer are generally able to suppress large excursions in microbial abundance and biomass. The key elements of such a control mechanism are size or surface-chemistry characteristics that link the dynamics of different microbial populations to common (nanoflagellate) predators and the fact that such predators are held well below their maximum growth rate potential at ambient food concentrations. This latter point, in particular, ensures a rapid and approximately linear increase in protistan growth and grazing pressure up to prey concentrations many times ambient levels. (C) 2002 Elsevier Science Ltd. All rights reserved.

Lavastre V, Girard D (2002) Tributyltin induces human neutrophil apoptosis and selective degradation of cytoskeletal proteins by caspases. J Toxicol Environ Health A 65 :1013-1024


Tributyltin (TBT) has frequently been used as a pesticide and in biocidal paints for marine vessels, leading to its presence in the environment. Although TBT was recently found to induce apoptosis in different immune cells, by a mechanism that is not fully established, its effect on neutrophils is not known. In this study, it was found that TBT induced apoptosis in human neutrophils as assessed by cytology, flow cytometry, and degradation of the microfilament-associated protein gelsolin. Furthermore, data showed that TBT induced neutrophil apoptosis by a caspase-dependent mechanism, since addition of z-Val-Ala-Asp(MOe)-CH(2)F (z-VAD-FMK) in the culture prevented the effect of TBT. It was also found that the cytoskeletal proteins gelsolin, paxillin, and vimentin, but not vinculin, were degraded by TBT via caspases, as assessed by immunoblotting. Data indicate that gelsolin, paxillin, and vimentin are three caspase substrates involved in both spontaneous and TBT-induced neutrophil apoptosis. Cells were not necrotic as assessed by trypan blue dye exclusion, and this is in agreement with the absence of vinculin degradation. Evidence indicates that TBT-induced fragmentation of cytoskeletal proteins via caspases is a process that is tightly regulated.

Lebaron P, Servais P, Baudoux AC, Bourrain M, Courties C, Parthuisot N (2002) Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry. Aquatic Microbial Ecology 28 :131-140

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In most aquatic bacterial communities, it is possible to discriminate bacterial cells with a high nucleic acid content (HNA) from those with a low nucleic acid content (LNA) by flow cytometry. The distribution of leucine incorporation rate per cell (specific activity) within the fraction of bacteria with a high apparent nucleic acid content was investigated in coastal seawater using flow cytometry and cell sorting techniques. Bacterial cells from a natural seawater sample were first labeled with tritiated leucine, stained with a fluorescent nucleic acid stain and then sorted based on their fluorescence and scatter signals, assuming that fluorescence was proportional to the cellular nucleic acid content and scatter signals to biovolume. Our results clearly demonstrated that specific activity was heterogeneously distributed within the group of HNA cells and increased with both scatter and fluorescence signals. This shows that important differences in terms of cell-specific rates of activity exist within the HNA group and that specific activity is positively correlated with both the nucleic acid content of cells and their biovolume, We hypothesized that this heterogeneity may be partly due to the structure of bacterial communities. Therefore, further investigations were made by analyzing the nucleic acid content and scatter properties of a set of 10 species isolated from the same coastal area. Our results confirm that both fluorescence and scatter values vary greatly between species at a given growth stage and between growth stages for a given species. Variations reported at different growth stages suggest that both parameters are sensitive to the metabolic activity of individual cells and confirm the positive relationship reported in this study between scatter and fluorescence parameters and specific activity within the HNA cellular fraction of natural communities. Interestingly, it was shown that HNA and LNA cells were present in late stationary phase cultures. This suggests that LNA cells found in natural communities may be dead or dying cells.

Ley V, Higgins J, Fayer R (2002) Bovine enteroviruses as indicators of fecal contamination. Applied and Environmental Microbiology 68 :3455-3461

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Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.

Li WK (2002) Macroecological patterns of phytoplankton in the northwestern North Atlantic Ocean. Nature 419 :154-157


Many issues in biological oceanography are regional or global in scope ; however, there are not many data sets of extensive areal coverage for marine plankton. In microbial ecology, a fruitful approach to large-scale questions is comparative analysis wherein statistical data patterns are sought from different ecosystems, frequently assembled from unrelated studies. A more recent approach termed macroecology characterizes phenomena emerging from large numbers of biological units by emphasizing the shapes and boundaries of statistical distributions, because these reflect the constraints on variation. Here, I use a set of flow cytometric measurements to provide macroecological perspectives on North Atlantic phytoplankton communities. Distinct trends of abundance in picophytoplankton and both small and large nanophytoplankton underlaid two patterns. First, total abundance of the three groups was related to assemblage mean-cell size according to the 3/4 power law of allometric scaling in biology. Second, cytometric diversity (an ataxonomic measure of assemblage entropy) was maximal at intermediate levels of water column stratification. Here, intermediate disturbance shapes diversity through an equitable distribution of cells in size classes, from which arises a high overall biomass. By subsuming local fluctuations, macroecology reveals meaningful patterns of phytoplankton at large scales.

Luna GM, Manini E, Danovaro R (2002) Large fraction of dead and inactive bacteria in coastal marine sediments : Comparison of protocols for determination and ecological significance. Applied and Environmental Microbiology 68 :3509-3513

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It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 +/- 0.2) x 10(8) cells g(-1) and (53.1 +/- 16.0) x 10(8) cells g(-1) in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count ; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was "reactivated." Bacterial turnover rates estimated ranged from 0.01 to 0.1 day(-1) but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day(-1)). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least I order of magnitude higher than previously reported.

Miura T, Ando N, Miura C, Yamauchi K (2002) Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica). Zoolog Sci 19 :321-329


In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitrol 1-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.

Not F, Simon N, Biegala IC, Vaulot D (2002) Application of fluorescent in situ hybridization coupled with tyramide signal amplification (FISH-TSA) to assess eukaryotic picoplankton composition. Aquatic Microbial Ecology 28 :157-166

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Photosynthetic picoeukaryotes (phytoplankton cells with a diameter smaller than 2 to 3 mum) contribute significantly to both biomass and primary production in the oligotrophic open ocean and coastal waters, at certain times of the year. The identification of these organisms is difficult because of their small size and simple morphology, therefore hindering detailed ecological studies of their distribution and role, In this paper, we demonstrate the use of oligonucleotide probes specific to algal classes or to lower order taxa in combination with fluorescent in situ hybridization and tyramide signal amplification (FISH-TSA) to determine eukaryotic picophytoplankton diversity, Target cells were detected and enumerated using epifluorescence microscopy. The sensitivity of the technique and the specificity of the probes were tested on pure and mixed picoplanktonic strains, as well as on natural samples from the English Channel. In these samples, the community was dominated by cells belonging to the division Chlorophyta. Haptophyta, Bolidophyceae and Pelagophyceae were also detected at low abundance. The FISH-TSA method is readily applicable to the study of picoplankton diversity in natural communities.

Peters F, Marrase C, Havskum H, Rassoulzadegan F, Dolan J, Alcaraz M, Gasol JM (2002) Turbulence and the microbial food web : effects on bacterial losses to predation and on community structure. Journal of Plankton Research 24 :321-331

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Changes in picoplankton population abundance and growth under turbulence have been suggested to be the consequence of turbulence affecting larger trophic levels and hence the grazing pressure. T I designed a laboratory set-up to assess the effects of turbulence on plankton assemblages, and tested the degree of food-web complexity needed to produce cascading effects on picoplankton and the interactions with nutrient enrichment. Grazing pressure on bacteria was relaxed under turbulence and we shou, that one trophic link is enough to produce effects at the picoplankton level. Nutrient enrichment increased the effect of turbulence as there was more biomass to act upon. The organisms responsible for driving the grazing pressure shifts could not be identified since they seemed to change depending on initial conditions and experimental treatment. A trend of increased heterotrophic biomass under turbulence it was found in all cases, which can have important implications in community metabolism dynamics.

Pillet S, Fournier M, Measures LN, Bouquegneau JM, Cyr DG (2002) Presence and regulation of metallothioneins in peripheral blood leukocytes of grey seals. Toxicol Appl Pharmacol 185 :207-217


Heavy metal residues have been reported at high levels in marine mammals. Although the immunotoxicity of these contaminants has been demonstrated in laboratory models, evaluation of their potential immunotoxicity for human and wildlife species is complicated by variables that modulate the immune response and the immunotoxic effects of xenobiotics under field conditions. Metallothioneins (MT) modulate the bioavaibility of physiological cations and the toxicity of heavy metals. Moreover, these proteins have been demonstrated to modulate immune functions. In the present study, we demonstrated a rapid and sustained increase of transcripts of the two major isoforms of MT (MT-1 and MT-2) in grey seal peripheral blood leukocytes (PBL) exposed in vitro to Zn. This increase in mRNA corresponds to an increase of MT proteins in PBL. However, we demonstrated a high heterogeneity among the three major PBL subpopulations (granulocytes, lymphocytes and monocytes). Monocytes appear to be the most sensitive cells to Zn or Cd exposure. Intracellular MT levels in PBL subpopulations were dependent not only on the duration of exposure and the concentration, but also on the metal species. Cd was more potent than Zn as an inducer of MT in lymphocytes but not in monocytes. Moreover, grey seal peripheral blood lymphocytes are less sensitive to Cd exposure than human lymphocytes. This noninvasive approach helps to better assess the risk of heavy metal exposure by considering the potential role of MT as modulators of immune response.

Qin QW, Ototake M, Nagoya H, Nakanishi T (2002) Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus. Vet Immunol Immunopathol 89 :83-89


The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.

Quintero-Betancourt W, Peele ER, Rose JB (2002) Cryptosporidium parvum and Cyclospora cayetanensis : a review of laboratory methods for detection of these waterborne parasites. J Microbiol Methods 49 :209-224


Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps.To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.

Rifa TG, Latatu A, Ayo B, Iriberri J, Comas-Riu J, Vives-Rego J (2002) Flow cytometric detection and quantification of heterotrophic nanoflagellates in enriched seawater and cultures. Systematic and Applied Microbiology 25 :100-108

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A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported. At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons : i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range. With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected. The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed. At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa. The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN : Rhynchomonas nasuta and Jakoba libera. Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms. In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.

Rioboo C, Gonzalez O, Herrero C, Cid A (2002) Physiological response of freshwater microalga (Chlorella vulgaris) to triazine and phenylurea herbicides. Aquat Toxicol 59 :225-235


The effects of two herbicides used wide-spread, isoproturon (phenylurea) and terbutryn (triazine), on growth, dry weight, elemental composition, photosynthetic pigments and protein content, and cell volume assayed by flow cytometry in the freshwater microalgae Chlorella vulgaris were studied. Different parameters for algal activity show widely different sensitivities to these aquatic pollutants. After 96 h of herbicide exposure, terbutryn was the strongest inhibitor of growth, giving an EC50 value for growth twice lower than that for isoproturon cultures (EC50 terbutryn=0.097 microM ; EC50 isoproturon=0.199 microM). However, lower concentrations of the triazine herbicide provoked an increase in the cellular density and growth rate of this microalga, not observed in the phenylurea-treated cultures. Cellular volume and dry weight of C. vulgaris cells were increased strongly in the presence of isoproturon and terbutryn. Other cellular parameters, such as pigment and protein content, were stimulated with both herbicides at higher concentrations.

Sasaki Y, Maita M, Okamoto N (2002) Rainbow trout neutrophils are responsible for non-specific cytotoxicity. Fish Shellfish Immunol 12 :243-252


This is the first report that rainbow trout (Oncorhynchus mykiss) neutrophils are responsible for non-specific cytotoxicity. A monoclonal antibody (mab) for rainbow trout leucocytes was produced. Using this mab (TTL-5E9), neutrophils (5E9-positive cells) were isolated from the pronephros by a panning technique. The isolated neutrophils showed high viability (approximately 95%) and purity (92-95%), and were functional in cytotoxic activity assays. The neutrophils demonstrated significantly higher cytotoxic activities against YAC-1 target cells than the other cells (5E9-negative cells, predominantly lymphocytes). The number of neutrophils contaminating the 5E9-negative fraction and their non-specific cytotoxicities were positively correlated. These findings demonstrate that rainbow trout neutrophils possess non-specific cytotoxic activities.

Sauve S, Brousseau P, Pellerin J, Morin Y, Senecal L, Goudreau P, Fournier M (2002) Phagocytic activity of marine and freshwater bivalves : in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn). Aquat Toxicol 58 :189-200


We measured non-specific immune function of various bivalves from marine (Cyrtodaria siliqua, Mactromeris polynyma, Mesosdesma arctatum, Mya arenaria, Mya truncata, Mytilus edulis, Serripes groenlandicus, Siliqua costata) and freshwater environments (Dreissena polymorpha and Elliptio complanata). We used flow cytometry to quantify the phagocytosis of fluorescent microspheres by hemocytes exposed in vitro to increasing levels of various metal compounds (AgNO(3), CdCl(2), CH(3)HgCl, HgCl(2) and ZnCl(2)). In some species, low doses of mercury (organic and inorganic) and Zn suggest a hormesis-like stimulation of phagocytic activity. At higher levels of exposure, all metals tested induced a significant dose-related inhibition of hemocyte phagocytosis. The species-specific sensitivity of the assay was determined by comparing the in vitro exposure using the metal concentration inducing a 50% suppression (EC(50)) of the phagocytic activity. Different species expressed different levels of sensitivity. Our results show the variability of the toxic response of different species within a group of similar organisms. It also highlights the need to consider species-species differences in ecotoxicological risk assessment.

Sauve S, Hendawi M, Brousseau P, Fournier M (2002) Phagocytic response of terrestrial and aquatic invertebrates following in vitro exposure to trace elements. Ecotoxicol Environ Saf 52 :21-29


The potential of the trace elements Ag, As, Cd, Hg, Mo, Ni, Pb, Se, and Zn to inhibit the phagocytosis response of extruded coelomocytes of different worm species was tested. We used flow cytometry to evaluate the sensitivity of cell viability and phagocytic potential for Eisenia fetida, Lumbricus terrestris, Aporrectodea turgida, and Tubifex tubifex. Extruded cells were exposed 18 h in vitro to concentrations ranging from 10(-9) to 10(-4) M. Mercury was the most potent immunotoxic element, with 50% inhibition of phagocytosis occurring at concentrations between 10(-7) and 10(-6) M. Cadmium, Cu, Ni, and Zn also showed significant immunosuppressive effects with concentrations inducing 50% inhibition ranging from 10(-5) to 10(-4) M. Species-specific sensitivity varied by about a factor of 10, with no species showing a systematically higher or lower in vitro sensitivity across the range of trace elements tested.

Scanlan DJ, West NJ (2002) Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus. Fems Microbiology Ecology 40 :1-12

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Oxygenic photoautotrophs of the genera Synechococcus and Prochlorococcus contribute significantly to primary production and are now widely accepted as the most abundant members of the picophytoplankton in the world’s oceans, Since they represent one of the Few cultured and representative groups of marine microorganisms, study of their physiology and biochemistry has progressed rapidly since their discovery. The recent and on-going sequencing of the complete genomes of representative strains will further hasten our understanding, and allow a complete interrogation, of the metabolism of these organisms, Moreover, since they inhabit a relatively simple environment they provide an excellent model system to begin to identify the underlying molecular mechanisms which allow their success in water columns with large vertical gradients of light and nutrients. Such work should provide novel insights into the genetic adaptations of these important marine microbes to their environment. We review here molecular ecological methods that are already available or which are currently being developed for these organisms. Such methods allow community structure. growth rate and nutrient status analysis, potentially at the single cell level, and can be used to define the niches, or identify the biotic or abiotic factors, which might control the productivity of specific genotypes. These techniques will undoubtedly provide the tools for answering more discerning questions concerning their ecology. How the complete genome sequence information is providing insights, and can further facilitate our understanding, of the ecology of these organisms is also discussed. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Seiler A, Visan A, Pohl I, Genschow E, Buesen R, Spielmann H (2002) Improving the embryonic stem cell test (EST) by establishing molecular endpoints of tissue specific development using murine embryonic stem cells (D3 cells). Altex-Alternativen Zu Tierexperimenten 19 :55-63

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Improving the embryonic stem cell test (EST) by establishing molecular endpoints of tissue specific development using murine embryonic stem cells (D3 cells).
Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. In the embryonic stem cell test (EST) the capacity of ES cells of the mouse cell line D3 to differentiate into contracting cardiomyocytes is used to assess the embryotoxic potential of test compounds and in addition, the effects on the viability of ES cells and differentiated mouse fibroblasts (cell line 3T3) are compared. The three endpoints are used to classify the embryotoxic potential of chemicals after 10 days of exposure : (i) the inhibition of differentiation of ES cells into cardiomyocytes (ID50) and (ii) the decrease of viability of 3T3 cells (IC(50)3T3) and (iii) ES cells (IC(50)D3) in a MTT cytotoxicity test. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study funded by ECVAM it could be demonstrated that the EST can predict the embryotoxic potential of a test compound as good as frequently used mammalian systems based on pregnant animals. In a joint project with major German pharmaceutical companies we are attempting to improve the EST by establishing molecular endpoints of differentiation (e.g. cardiac, neuronal, chondrogenic) in cultured ES cells. We have studied the expression of tissue specific proteins in ES cell cultures in the presence of embryotoxic chemicals by immunofluorescent antibody techniques, e.g. FACS analysis. The other groups are focusing on endogenous gene expression in early development by RT-PCR methods or the DNA microarray technique. The results obtained recently using molecular markers specific for cardiac differentiation and employing intracellular flow cytometry for quantification will be presented. Molecular endpoints will allow improvement of the EST by measuring gene expression patterns in a small number of marine ES cells.

Serra T, Casamitjana X, Colomer J, Granata TC (2002) Observations of the particle size distribution and concentration in a coastal system using an in situ laser analyzer. Marine Technology Society Journal 36 :59-69

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An in situ laser particle size analyzer (LISST-100, Sequoia Scientific, Inc.) has been used to study the particle size distribution and concentration of biological and non biological particles in the water column of a Mediterranean coastal system. Two field campaigns have been carried out during low and high energy conditions of the flow, caused by the passage of a storm front. For the low energy period, the water column remained stratified, whereas for the high energetic period the water column was warmer and well mixed. The first study dealt with the distribution of particles near the bottom of the coastal area. Here, two regions were taken into account. The first region was a seagrass meadow of Posidonia oceanica and the second region was a barren sand area. ne second study dealt with the determination of the vertical distribution of suspended particles in the whole water column of the system. Me results showed a decrease in the vertical concentration of suspended particles in the water column with the passage of the storm front, which was associated with advection of warm water mass rather than by vertical mixing. In contrast, vertical resuspension determined the fate of suspended particles at the bottom of the water column and an increase of their concentration was found.

Sheehy MRJ (2002) A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches. Archives of Gerontology and Geriatrics 34 :233-248

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The ability to measure lipofuscin accumulation accurately is essential for understanding its role in physiological ageing and human disease, and for its recent use as an ecological tool for age determination. Existing quantification methods are problematic. In situ histological measurement by microscopy can be very precise but is labour intensive. Spectrofluorimetric measurement of whole lipid extracts is rapid but not sufficiently specific. A recent HPLC assay for the retinal pigment epithelium lipofuscin fluorophore, A2-E is potentially both precise and rapid but not applicable to lipofuscin in other tissues, or from fixed samples. In this study, I explore the use of flow cytometry or fluorescence activated cell sorting (FACS) for specific quantification of lipofuscin granules in formalin-fixed CNS homogenates from lobsters (Homarus gammarus). Free neurolipofuscin grannles were discriminated in FACS samples by their size distribution (forward scatter), distinctive orange autofluorescence (FL3) and refractive internal structure (side scatter). A quantitative neurolipofuscin index was developed, which was highly correlated with the microscopically measured neurolipofuscin concentration in the same tissue. Sample-processing rate was at least an order of magnitude eater for FACS than for quantitative microscopy but the latter yielded a much more precise estimate of neurolipofuscin concentration. While the FACS approach may be ideal where rapid handling and only semiquantitative results are required, loss of precision will preclude use in many ecological studies where the highest available resolution is needed. Further refinements to the FACS approach are possible but advanced histological methods for neurolipofuscin quantification remain the most reliable at this time. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Sherr EB, Sherr BF (2002) Significance of predation by protists in aquatic microbial food webs. Antonie Van Leeuwenhoek 81 :293-308


Predation in aquatic microbial food webs is dominated by phagotrophic protists, yet these microorganisms are still understudied compared to bacteria and phytoplankton. In pelagic ecosystems, predaceous protists are ubiquitous, range in size from 2 gm flagellates to > 100 microm ciliates and dinoflagellates, and exhibit a wide array of feeding strategies. Their trophic states run the gamut from strictly phagotrophic, to mixotrophic : partly autotrophic and partly phagotrophic, to primarily autotrophic but capable of phagotrophy. Protists are a major source of mortality for both heterotrophic and autotrophic bacteria. They compete with herbivorous meso- and macro-zooplankton for all size classes of phytoplankton. Protist grazing may affect the rate of organic sinking flux from the euphotic zone. Protist excretions are an important source of remineralized nutrients, and of colloidal and dissolved trace metals such as iron, in aquatic systems. Work on predation by protists is being facilitated by methodological advances, e.g., molecular genetic analysis of protistan diversity and application of flow cytometry to study population growth and feeding rates. Examples of new research areas are studies of impact of protistan predation on the community structure of prey assemblages and of chemical communication between predator and prey in microbial food webs.

Simon N, Biegala IC, Smith EA, Vaulot D (2002) Kinetics of attachment of potentially toxic bacteria to Alexandrium tamarense. Aquatic Microbial Ecology 28 :249-256

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Interactions between bacteria and harmful algae are potentially important regulating factors for population dynamics of both organisms and for toxin production. These interactions are still poorly understood. To monitor the physical associations between potentially toxic bacteria and dinoflagellates in controlled conditions, we inoculated an axenic non-toxic strain of Alexandrium tamarense (Dinophyceae) together with reputed paralytic shellfish toxin (PST)-producing bacteria belonging to the genus Alteromonas (gamma subdivision of the division Proteobacteria) and to the clade Roseobacter (alpha subdivision of the division Proteobacteria). The attachment behavior of both bacterial strains was monitored using TSA-FISH (tyramide signal amplification and fluorescent in situ hybridization) and confocal microscopy. Our results suggest that ageing dinoflagellate cultures stimulate both free bacterial growth and attachment. However, toxin production by originally non-toxic dinoflagellate cells was not induced by the physical interaction of either of the bacterial strains with the dinoflagellate cells. This does not support the hypothesis that toxic bacteria could simply control toxin production by attachment to particle surfaces such as eukaryotic organisms.

Sosik HM, Olson RJ (2002) Phytoplankton and iron limitation of photosynthetic efficiency in the Southern Ocean during late summer. Deep-Sea Research Part I-Oceanographic Research Papers 49 :1195-1216

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As part of two USJGOFS cruises, we investigated spatial variability in phytoplankton properties across the strong environmental gradient associated with the Antarctic Polar Frontal Zone during late austral summers of 1997 and 1998. Cell properties, including size and an index of pigment content as well as photosynthetic efficiency (as indicated by relative variable fluorescence), changed dramatically across this frontal region. A general trend toward reduced photosynthetic efficiency south of the Polar Front was correlated with low dissolved iron concentration and is consistent with physiological iron limitation in the phytoplankton. We detected no significant differences in photosynthetic efficiency among different size classes of the dominant pico- to nanophytoplankton, despite a systematic community level shift toward larger sized cells south of the Polar Front. In contrast to other cells, those classified as cryptophyte algae showed relatively high photosynthetic efficiency in low iron waters ; however, this group was never found in high abundance. One group, all cells less than or equal to 2 mum, showed an unexpected increase in intracellular pigment content (based on single cell chlorophyll fluorescence measurements) south of the Polar Front where dissolved iron concentration and the cells’ relative abundance were low. Overall, these results suggest that group- or size-specific differences in physiological status were not directly regulating community structure in the pico- to nanophytoplankton during the late summer season ; other processes, such as differential grazing or sinking losses, must be important. (C) 2002 Published by Elsevier Science Ltd.

Stauber JL, Franklin NM, Adams MS (2002) Applications of flow cytometry to ecotoxicity testing using microalgae. Trends Biotechnol 20 :141-143


Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.

Stoderegger KE, Herndl GJ (2002) Distribution of capsulated bacterioplankton in the North Atlantic and North Sea. Microbial Ecology 44 :154-163

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In laboratory experiments, bacterioplankton were incubated under different nutrient conditions, and the percentage of bacteria exhibiting a polysaccharidic capsule (capsulated bacteria) and that of CTC (cyanotetrazolium chloride) -positive and therefore metabolically highly active bacteria were determined. In these seawater cultures amended with nutrients more than 95% of the CTC-positive cells exhibited a capsule. During two cruises, one to the North Atlantic and one to the North Sea, we investigated the distribution of capsulated bacteria throughout the water column. Capsulated bacteria were generally more abundant in eutrophic surface waters than in deeper layers or more oligotrophic regions. In the upper 100 m of the North Atlantic, about 6-14% of the total bacterioplankton community was capsulated, while in the layers below 100 m depth, 97% of the bacteria lacked a visible capsule. The percentage of capsulated bacteria correlated with bacterial abundance and production, and chlorophyll a concentration. Also, the bioavailability of DOC (dissolved organic carbon), estimated by the ratio between bacterial production and DOC concentration, significantly correlated with the percentage of capsulated bacteria. In the North Sea, the contribution of capsulated bacteria to the total number of bacteria decreased from the surface (3 m depth) to the near-bottom (25-35 m) layers from 20% to 14% capsulated bacteria. In the nearshore area of the North Sea, about 27% of the bacteria exhibited a capsule. Overall, a pronounced decrease in the contribution of capsulated bacteria to the total bacterial abundance was detectable from the eutrophic coastal environment to the open North Atlantic. Using this epifluorescence-based technique to enumerate capsulated bacterioplankton thus allowed us to routinely assess the number of capsulated bacteria even in the oceanic water column. Based on the data obtained in this study we conclude that almost all metabolically highly active bacteria exhibit a capsule, but also some of the metabolically less active cells express a polysaccharide capsule detectable with this method.

Takeuchi Y, Yoshizaki G, Kobayashi T, Takeuchi T (2002) Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter. Biol Reprod 67 :1087-1092


Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.

Thyrhaug R, Larsen A, Brussaard CPD, Bratbak G (2002) Cell cycle dependent virus production in marine phytoplankton. Journal of Phycology 38 :338-343

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dIn this study we investigated virus production in two marine phytoplankton species and how it relates to the host’s cell cycle. Phaeocystis pouchetii (Hariot) Lagerheim and Pyramimonas orientalis McFadden, Hill & Wetherby, growing synchronously in batch cultures, were infected with their respective viruses (PpV and PoV) at four different stages in the cell cycle and the production of free virus was then measured for 30 h. The virus production in P. orientalis infected with PoV depended on the time of infection, whereas no such relation was found for P. pouchetii infected with PpV. The P. orientalis cultures infected at the end of the dark period and at the beginning of the light period produced three times more virus than those infected in the middle of the light period and eight times more virus than those infected at the beginning of the dark period. The latent periods for PpV and PoV were 12-14 h and 18-20 h, respectively, and in both cases were independent of the host cell cycle. The differences in virus production may be attributed to light or cell cycle dependent regulation of host infection, metabolism, or burst size. Regardless of the mechanism, these differences maybe related to differences in the ecological strategies of the hosts and their ability to form blooms.

Troussellier M, Schafer H, Batailler N, Bernard L, Courties C, Lebaron P, Muyzer G, Servais P, Vives-Rego J (2002) Bacterial activity and genetic richness along an estuarine gradient (Rhone River plume, France). Aquatic Microbial Ecology 28 :13-24

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Bacterial diversity and activity were simultaneously investigated by microbial ecological and molecular biological methods along an estuarine gradient from the Rhone River to the Mediterranean Sea. Following a Lagrangian strategy, we sampled plume, frontal and marine layers. The sampled estuarine gradient exhibited large changes both in physico-chemical and in microbiological characteristics. Bacterial abundances and activities showed a more drastic decrease in the low salinity range of the gradient than expected from simple dilution models, indicating that an important fraction of freshwater bacteria disappeared in the mixing area, High specific activities, in particular for leucine, in the marine end-part of the gradient suggested important bacterial protein synthesis, which may be a sign of an active survival strategy for bacterial communities subjected to oligotrophic conditions. Bacterial genetic diversity of the sampled estuarine area, as estimated by the number of DNA-derived denaturing gradient gel electrophoresis (DGGE) bands, was high (13 to 55 bands) compared to that reported in other aquatic ecosystems. This high diversity may be the consequence of the interface position of estuaries. The proportion of active populations was estimated using the ratio of DGGE bands derived from RNA and DNA. This ratio was lower in Rhone water than in marine water, indicating that only a part of the constitutive populations were active, while the activity was distributed within a larger fraction Of populations in the marine assemblage, Very few DGGE bands detected in freshwater samples were also detected in the marine end-part of the gradient, suggesting that a very limited number of freshwater bacteria could survive under marine conditions. Detection of these freshwater populations from RNA might indicate that these bacteria were able to synthesize different stress proteins as the result of a survival strategy or that these bacteria were able to maintain metabolic activity under marine conditions, The structure of marine communities was strongly affected by decreasing salinity. However, it seems that the decrease of DNA-derived bands may simply have been the consequence of the mixing of marine and freshwater. No obvious relationship between genetic richness and activity changes was observed. This lack of a relationship may be the consequence of a very short residence time of water in the mixing area studied.

Vardy S, Uwins P (2002) Fourier transform infrared microspectroscopy as a tool to differentiate Nitzschia closterium and Nitzschia longissima. Applied Spectroscopy 56 :1545-1548

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Nitzschia closterium and Nitzschia longissima are two species of diatom that are extremely difficult to differentiate using light microscopy. This paper details an investigation into the use of FT-IR microscopy combined with discriminant analysis to differentiate between these species. Spectra were taken from unidentified samples and classified against a training set using either Mahalanobis distances or principal component analysis combined with canonical discriminant analysis. Unidentified samples were classified with a 100% accuracy using both mathematical techniques.

Vila X, Guyoneaud R, Cristina XP, Figueras JB, Abella CA (2002) Green sulfur bacteria from hypersaline Chiprana Lake (Monegros, Spain) : habitat description and phylogenetic relationship of isolated strains. Photosynthesis Research 71 :165-172

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The ’Salada de Chiprana’ (Chiprana Lake) is a hypersaline (30-73parts per thousand), permanent and shallow lake of endorheic origin in a semi-arid region of the Ebro depression (Aragon, Spain). Magnesium sulfate and sodium chloride represent the main salts of this athalassohaline environment. Anoxic conditions occurred periodically in the bottom layers of the lake during the study period. When stratified, high sulfide concentrations (up to 7 mM) were measured in the hypolimnion. Physical and chemical conditions gave rise to the development of very dense green sulfur bacteria blooms (10.7 mg l(-1) of BChl c and 16.7 mor l(-1) of BChl d) at 0.5-1 m from the bottom. Microscopic observations revealed that cells morphologically similar to Chlorobium vibrioforme were dominant in the phototrophic bacterial community, but Prosthecochloris aestuarii was also found sometimes at lower concentrations, as revealed by both microscopic observation and flow cytometric analyses. Deep agar dilution series allowed to obtain several axenic cultures of phototrophic bacteria. They were identified according to their morphology, pigment composition and phylogenetic relationships (16S rDNA sequence analysis). Two of the sequenced strains (CHP3401 and CHP3402) belonged to the green sulfur bacteria and were related to Prosthecochloris aestuarii SK413(T) and Chlorobium vibrioforme DSM260(T), respectively. HPLC analyses of both natural samples and Chlorobium vibrioforme isolates indicated that these strains contained both BChl c and BChl d. Phylogenetic results suggested that Chlorobium vibrioforme strains DSM260(T) and CHP3402, all sequenced strains of Prosthecochloris aestuarii and strain CIB2401 constitute a separate cluster of green sulfur bacteria, all of them isolated from marine to hypersaline habitats.

Volkman JK, Tanoue E (2002) Chemical and biological studies of particulate organic matter in the ocean. Journal of Oceanography 58 :265-279

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This paper presents a review of the past decade’s highlights of research on the isolation and characterisation of particulate organic matter (POM) in the world’s oceans. The emphasis is on chemical studies but, in keeping with the growing interdisciplinary nature of marine science, advances in other disciplines are also discussed, particularly those in biological sciences. Increasing evidence for the importance of picoplankton, bacteria and viruses as POM constituents is highlighted, including the recent recognition of large populations of autotrophic bacteria able to harvest light for energy. The transport of POM to bottom waters was thought to be largely confined to large, rapidly sinking faecal pellets. However, recent studies have highlighted the importance of organic aggregates and floes formed by diatoms such as Rhizosolenia and other microalgae. Ascending particles have also been discovered, many of which are lipid-rich. Several studies have shown that resuspension of bottom sediments and lateral advection of material from continental shelves can lead to anomalously high particle fluxes measured in sediment traps moored in deep water. Many new approaches for characterizing POM have emerged, such as pyrolysis gas chromatography-mass spectrometry and direct temperature-resolved mass spectrometry for analysis of higher molecular weight materials and biopolymers. Lipid biomarker techniques have also advanced, exciting new possibilities being raised by the ability to measure stable and radioactive carbon isotopes for individual compounds. The techniques of molecular biology, such as the polymerase chain reaction (PCR), are being increasingly applied to provide complementary information to more conventional microscopy and flow cytometry on the identity of organisms in the sea. The combination of these techniques with advanced chemical analysis methods promises to greatly increase our knowledge of the origins, transport and fate of organic matter in the oceans.

Waterhouse R, Ha C, Dveksler GS (2002) Murine CD9 is the receptor for pregnancy-specific glycoprotein 17. Journal of Experimental Medicine 195 :277-282

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Pregnancy-specific glycoproteins (PSGs) are a family of highly similar secreted proteins produced by the placenta. PSG homologs have been identified in primates and rodents. Members of the human and murine PSG family induce secretion of antiinflammatory cytokines in mononuclear phagocytes. For the purpose of cloning the receptor, we screened a RAW 264.7 cell cDNA expression library. The PSG17 receptor was identified as the tetraspanin, CD9. We confirmed binding of PSG17 to CD9 by ELISA, flow cytometry, alkaline phosphatase binding assays, and in situ rosetting. Anti-CD9 monoclonal antibody inhibited binding of PSG17 to CD9-transfected cells and PAW 264.7 cells. Moreover, PSG 17 binding to macrophages front CD9-deficient mice was significantly reduced. We theta tested whether PSG 17 binds to other members of the marine tetraspanin family. PSG17 did not bind to cells transfected with CD53, CD63, CD81, CD82, or CD151, suggesting that PSG17-CD9 binding is a specific interaction. We have identified the first receptor for a murine PSG as well as the first natural ligand for a member of the tetraspanin superfamily.

Wawrik B, Paul JH, Tabita FR (2002) Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl Environ Microbiol 68 :3771-3779


Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional 35S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95 ; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29 ; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.

Weisse T (2002) The significance of inter- and intraspecific variation in bacterivorous and herbivorous protists. Antonie Van Leeuwenhoek 81 :327-341


This paper reviews the emerging evidence on the significance of inter- and intraspecific variation in the feeding behaviour of aquatic protists. Small heterotrophic nanoflagellates (HNF) have been identified as the primary bacterial consumers in most aquatic environments. Recent research using novel techniques such as flow cytometry and high resolution video microscopy revealed that their feeding strategies and grazing rates are diverse. There is an important conceptual difference between uptake rates measured in short-term (min to h) experiments and grazing rates averaged over a longer-term (d). This is because the latter are strongly affected by digestion rates which are species-specific, i.e. the same bacterial prey may be digested differently by various grazers, and the same predator may selectively digest variable prey. Planktonic ciliates are the most important algal consumers in many lakes and marine systems. Large species-specific differences in their feeding behaviour and growth rates have been documented for closely related species. Intraspecific variation, which is, most likely, caused by varying clonal composition may be as important as interspecific variation. Finally, there is some evidence that the individual variability within a given population is generally large, both among bacterivorous HNF and among herbivorous ciliates. The consequences of this diversity becoming apparent at the levels of the species, population, clone and individual need to be considered by aquatic ecologists in their conceptual models.

Wilson WH, Tarran G, Zubkov MV (2002) Virus dynamics in a coccolithophore-dominated bloom in the North Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography 49 :2951-2963

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We used analytical flow cytometry (AFC) to determine virus concentrations through vertical profiles in a coccolithophore-dominated bloom in the northern North Sea during June 1999. We present the first high-intensity sampling data of viruses from a lagrangian survey to gain a unique insight into the temporal and spatial dynamics of viruses in an open-water sight. Virus abundances ranged from 2.6 x 10(5) to 5.4 x 10(6) ml(-1), which is within the range expected for open-water environments. The highest concentrations were invariably observed in surface waters. During the lagrangian experiment there was a net decrease in virus numbers, suggesting that they were actively infecting hosts. Large viruses could be easily discriminated from small viruses since there was at least an order of magnitude difference in their AFC side-scatter values. Large viruses, assumed to infect DMS-producing algae, did not appear to influence DMS/DMSP production. It is likely that microzooplankton out-competed viruses for coccolithophore prey/hosts. Lower small virus to bacteria ratios (VBR) were observed in a subsurface layer compared to the more productive surface layers. The subsurface layer was dominated by a species of alpha-proteobacteria related to the genus Roseobacter, and the low VBR may indicate that viruses were infecting Roseobacter in this layer. Application of AFC is an excellent technique for high-definition sampling of virus communities, although it is recognised that we are working at the limit of detection for many small viruses using currently available nucleic acid stains. (C) 2002 Elsevier Science Ltd. All rights reserved.

Wilson WH, Tarran GA, Schroeder D, Cox M, Oke J, Malin G (2002) Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel. Journal of the Marine Biological Association of the United Kingdom 82 :369-377

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This study used analytical flow cytometry (AFC) to monitor the abundance of phytoplankton, coccoliths, bacteria and viruses in a transect that crossed a high reflectance area in the western English Channel. The high reflectance area, observed by satellite, was caused by the demise of an Emiliania huxleyi bloom. Water samples were collected from depth profiles at four stations, one station Outside and three stations inside the high reflectance area. Plots of transect data revealed very obvious differences between Station 1, outside, and Stations 2-4, inside the high reflectance area. Inside, concentrations of viruses were higher ; E. huxleyi cells were lower ; coccoliths were higher ; bacteria were higher and virus:bacteria ratio was lower than at Station 1, outside the high reflectance area. This data can simply be interpreted as virus-induced lysis of E. huxleyi cells in the bloom causing large concentrations of coccoliths to detach, resulting in the high reflectance observed by satellite imagery. This interpretation was supported by the isolation of two viruses, EhV84 and EhV86, from the high reflectance area that lysed cultures of E. huxleyi host strain CCMP1516. Basic characterization revealed that they were lytic viruses approximately 170 nm -190 nm in diameter with an icosahedral symmetry. Taken together, transect and isolation data suggest that Viruses were the major contributor to the demise of the E. huxleyi population in the high reflectance area. Close coupling between microalgae, bacteria and viruses contributed to a large organic carbon input. Consequent cycling influenced the succession of an E. huxleyi-dominated population to a more characteristic mixed summer phytoplankton community.

Wirsen CO, Sievert SM, Cavanaugh CM, Molyneaux SJ, Ahmad A, Taylor LT, DeLong EF, Taylor CD (2002) Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp that produces filamentous sulfur. Applied and Environmental Microbiology 68 :316-325

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A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4’,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Wood AM, Miller SR, Li WKW, Castenholz RW (2002) Preliminary studies of cyanobacteria, picoplankton, and virioplankton in the Salton Sea with special attention to phylogenetic diversity among eight strains of filamentous cyanobacteria. Hydrobiologia 473 :77-92

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Cyanobacterial diversity in the Salton Sea, a high-salinity, eutrophic lake in Southern California, was investigated using a combination of molecular and morphological approaches. Representatives of a total of 10 described genera (Oscillatoria, Spirulina, Arthrospira, Geitlerinema, Lyngbya, Leptolyngbya, Calothrix, Rivularia, Synechococcus, Synechocystis) were identified in the samples ; additionally, the morphology of two cultured strains do not conform to any genus recognized at present by the bacteriological system. Genetic analysis, based on partial 16S rRNA sequences suggested considerable cryptic genetic variability among filamentous strains of similar or identical morphology and showed members of the form-genus Geitlerinema to be distributed among three major phylogenetic clades of cyanobacteria. Cyanobacterial mats, previously described from the Sea were, in fact, composed of both filamentous cyanobacteria and a roughly equivalent biomass of the sulfur-oxidizing bacterium Beggiatoa, indicating their formation in sulfide rich regions of the lake. Flow cytometric analysis of the water samples showed three striking differences between samples from the Salton Sea and representative marine waters : (1) phycoerythrin-containing unicells, while abundant, were much less abundant in the Salton Sea than they were in typical continental shelf waters, (2) Prochlorococcus appears to be completely absent, and (3) small (3-5 mum) eukaryotic algae were more abundant in the Salton Sea than in typical neritic waters by one-to-two orders-of-magnitude. Based on flow cytometric analysis, heterotrophic bacteria were more than an order of magnitude more abundant in the Salton Sea than in seawater collected from continental shelf environments. Virus particles were more abundant in the Salton Sea than in typical neritic waters, but did not show increases proportionate with the increase in bacteria, picocyanobacteria, or eukaryotic algae.

Zubkov MV, Fuchs BM, Archer SD, Kiene RP, Amann R, Burkill PH (2002) Rapid turnover of dissolved DMS and DMSP by defined bacterioplankton communities in the stratified euphotic zone of the North Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography 49 :3017-3038

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Bacterioplankton-driven turnover of the algal osmolyte, dimethylsulphoniopropionate (DMSP), and its degradation product, dimethylsulphide (DMS) the major natural source of atmospheric sulphur, were studied during a Lagrangian SF6-tracer experiment in the North Sea (60degreesN, YE). The water mass sampled within the euphotic zone was characterised by a surface mixed layer (from 0 m to 13-30 m) and a subsurface layer (from 13-30 m to 45-58 m) separated by a 2degreesC thermocline spanning 2m. The fluxes of dissolved DMSP (DMSPd) and DMS were determined using radioactive tracer techniques. Rates of the simultaneous incorporation of C-14-leucine and H-3-thymidine were measured to estimate bacterioplankton production. Flow cytometry was employed to discriminate subpopulations and to determine the numbers and biomass of bacterioplankton by staining for nucleic acids and proteins. Bacterioplankton subpopulations were separated by flow cytometric sorting and their composition determined using 16S ribosomal gene cloning/sequencing and fluorescence in situ hybridisation with designed group-specific oligonucleotide probes. A subpopulation, dominated by bacteria related to Roseobacter-(alpha-proteobacteria), constituted 26-33% of total bacterioplankton numbers and 45-48% (of biomass in both surface and subsurface layers. The other abundant prokaryotes were a group within the SAR86 cluster of gamma-proteobacteria and bacteria from the Cytophaga-Flavobacterium-cluster. Bacterial consumption of DMSPd was greater in the subsurface layer (41 nM d(-1)) than in the surface layer (20 nM d(-1)). Bacterioplankton tightly. controlled the DMSPd pool, particularly in the subsurface layer, with a turnover time of 2 h, whereas the turnover time of DMSPd in the surface layer was 10 h. Consumed DMSP satisfied the majority of sulphur demands of bacterioplankton, even though bacterioplankton assimilated only about 2.5% and 6.0% of consumed DMSPd sulphur in the surface and subsurface layers, respectively. Bacterioplankton turnover of DMS was also faster in the subsurface layer (12 h) compared to the surface layer (24 h). However, absolute DMS consumption rates were higher in the surface layer, due to higher DMS concentrations in this layer. The majority of DMS was metabolised into dissolved non-volatile products, and bacteria could satisfy only 1-3% of their sulphur demands from DMS. Thus, structurally similar bacterioplankton communities exerted strong control over DMSPd and DMS concentrations both in the subsurface layer and surface mixed layer. (C) 2002 Elsevier Science Ltd. All rights reserved.

Zubkov MV, Fuchs BM, Tarran GA, Burkill PH, Amann R (2002) Mesoscale distribution of dominant bacterioplankton groups in the northern North Sea in early summer. Aquatic Microbial Ecology 29 :135-144

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A case study was performed to evaluate the potential of a combination of flow cytometry, cell sorting and fluorescence in situ hybridisation (FISH) for mesoscale monitoring of dominant bacterioplankton groups. In June 1999, the spatial distribution of phylogenetically characterised bacterioplankton groups in an area of 150 x 350 km in the northern North Sea was studied in conjunction with blooms of phytoplankton including coccolithophores. The bacterial cells were enumerated using flow cytometry and 3 groups were defined on the basis of cellular light scatter, nucleic acid and protein content. The phylogenetic composition of cells sorted from those 3 groups was analysed using FISH with a restricted set of rRNA targeted oligonucleotide probes. Cells with high nucleic acid and high protein content were mainly affiliated to the alpha-proteobacterial genus Roseobacter. Members of the Cytophaga-Flavobacterium cluster consistently accounted for the majority of cells in the high nucleic acid and low protein group ; and members of the gamma-proteobacterial SAR86 cluster were always present in significant amounts among the cells with low nucleic acid and low protein content. The composition of bacteria within the groups was remarkably conservative in 13 randomly selected samples, while the concentration of the groups varied considerably on the 10 to 100 km scale. The bacterial groups formed patches of high abundance ; these were spatially separated and could remain traceable for 2 to 3 d. The distribution of bacterioplankton groups did not correlate with distribution of either chlorophyll a (chl a), or phytoplankton groups (small coccolithophores and picoeukaryotic algae) or physical parameters such as temperature and salinity. It seems unlikely therefore that currently used remotely sensed parameters may be used as proxies of bacterioplankton group concentration at basin scales. However, this case study proves that a shipboard survey conducted over 4 to 6 d is effective for identifying and monitoring patches of certain bacterioplankton groups.