mardi 21 avril 2009
par   G. Grégori

(2001) Flow cytometry in the marine environment. Proceedings from the 20th ISAC Congress. Montpellier, France, 20-25 May 2000. Cytometry 44 :163-271


Andreatta S, Wallinger MM, Posch T, Psenner R (2001) Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis. Cytometry 44 :218-225


BACKGROUND : Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS : A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS : Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS : Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.

Babichenko S, Leeben A, Poryvkina L (2001) Variability of Chlorella sp fluorescence in response to different nitrogen conditions. International Journal of Remote Sensing 22 :403-414

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The variability of quantitative fluorescence characteristics of Chlorella sp. (strain TV217) pigments during culture growth under different nitrogen conditions was studied by using excitation spectra of in vivo fluorescence. The ratio of chlorophyll-b/chlorophyll-a, calculated for both concentration and fluorescence levels, was stable for the selected culture and depended weakly on the environmental conditions and growth phase. However, the relative fluorescence efficiency of chlorophylls and chlorophyll-a-specific fluorescence in culture with inorganic additions showed the lowest values at the exponential phase of growth. Chlorophyll-a-specific fluorescence, investigated in field experiments on natural phytoplankton, showed a similar pattern. Generally, the chlorophyll-a-specific fluorescence was two to five times less during intensive growth of the community compared with the stationary stage. The possibilities of using fluorescence for quantitative calibration of phytoplankton pigment content are discussed.

Benfey TJ (2001) Use of sterile triploid Atlantic salmon (Salmo salar L.) for aquaculture in New Brunswick, Canada. Ices Journal of Marine Science 58 :525-529

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Induced triploidy is the only effective method currently available for mass production of reproductively sterile salmonids fbr aquaculture. Repeated studies at the Atlantic Salmon Federation’s hatchery (St Andrews, New Brunswick. Canada) hac e shown only minor differences between triploids and diploids : in survival to S1 smolt agr (15 months), percentage of the population which became S1 smolts. and mean S1 smelt size. However. a similar study at a commercial hatchery was terminated because of exceptionally high mortality of triploids prior to the start of feeding. Marine growout trials in sea cages showed that triploids grew well in seawater. but had reduced survival rates (leading to a 5-15% reduction in yield at harvest) and high rates of jaw abnormalities. Similar results have been reported elsewhere. Although induced triploidy can be used effectively as a management tool to ensure lack of reproduction. there is at present little support of the aquaculture industry to switch to their large-scale use. In light of fundamental biological differences, it is perhaps naive to expect triploids to perform as well as diploids using standard culture methods. Triploids should be treated as a new "species" For aquaculture development. beginning with research to determine their optimum rearing requirements, (C) 2001 International Council for the Exploration of the Sea.

Berman T, Kaplan B, Chava S, Viner Y, Sherr BF, Sherr EB (2001) Metabolically active bacteria in Lake Kinneret. Aquatic Microbial Ecology 23 :213-224

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Three staining methods were used to identify metabolically active bacteria in Lake Kinneret, northern Israel : CTC, DAPI staining followed by a propanol wash, and the Molecular Probes Live/Dead stain. Positive results from these methods purport to show, respectively, actively respiring bacteria (CTC+), cells with intact nucleoids (NuCC), and cells with intact membranes (MEM+). Concomitantly, bacterial metabolic activity was measured as electron transport system (ETS) flux, O-2 uptake, activities of peptidase, beta -glucosidase and Lipase, and rate of leucine incorporation in monthly samples taken for 2.5 yr at a pelagic lake station. Laboratory experiments followed changes during 22 or 40 h in the percentages of ’active’ bacteria in GF/C-filtered lake water with or without substrate enrichment or antibiotic inhibitors of cell division, or with bacterivorous protists. In lake samples, each of the staining methods detected different aspects of cellular state or metabolic activity but all 3 indicated low percentages of ’active’ bacteria relative to total bacterial abundance. CTC+ ranged from 1.0 to 27.3% (average 5.1%), NuCC from 1.4 to 42.9% (average 8.3 %) and MEM+ from 1.0 to 29.9 % (average 8.8 %), with no clear seasonal or spatial patterns. No significant correlations were found between the proportions of ’active’ bacteria in lake water as determined by these methods, although such correlations were observed in the laboratory experiments. Significant correlations were obtained between ETS and O-2 uptake, peptidase and P-glucosidase, and between leucine incorporation and peptidase. ETS was significantly correlated with CTC+ and NuCC cell abundance, but not with total bacteria (DAPI counts). In contrast, peptidase activity correlated with total bacterial counts. Results of time course experiments indicated that some bacteria which initially appear to be inactive can become active when stimulated by substrate addition, even though cell division is inhibited. Grazing by protists increased the percentage of active bacteria, at least during the active predator-prey phase. Our data support the hypothesis that in natural waters usually only a small fraction (probably <20 %) of the entire bacterial assemblage is strongly active metabolically at any given time. This proportion may increase dramatically with localized substrate inputs. The concept of bacterial assemblages, heterogeneous not only in terms of phylotype, but also in terms of levels of metabolic activity will need to be considered in future aquatic ecosystem models.

Bernard L, Courties C, Duperray C, Schafer H, Muyzer G, Lebaron P (2001) A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems. Cytometry 43 :314-321


BACKGROUND : Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS : Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS : The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION : This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

Blanchot J, Andre JM, Navarette C, Neveux J, Radenac MH (2001) Picophytoplankton in the equatorial Pacific : vertical distributions in the warm pool and in the high nutrient low chlorophyll conditions. Deep-Sea Research Part I-Oceanographic Research Papers 48 :297-314

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Abundance distribution and cellular characteristics of picophytoplankton were studied in two distinct regions of the equatorial Pacific : the western warm pool (0 degrees, 167 degreesE), where oligotrophic conditions prevail, and the equatorial upwelling at 150 degreesW characterized by high-nutrient low-chlorophyll (HNLC) conditions. The study was done in September-October 1994 during abnormally warm conditions. Populations of Prochlorococcus, orange fluorescing Synechococcus and picoeukaryotes were enumerated by flow cytometry. Pigment concentrations were studied by spectrofluorometry. In the warm pool, Prochlorococcus were clearly the dominant organisms in terms of cell abundance, estimated carbon biomass and measured pigment concentration. Integrated concentrations of Prochlorococcus, Synechococcus and picoeukaryotes were 1.5x10(13), 1.3x10(11) and 1.5x10(11)cells m(-2), respectively. Integrated estimated carbon biomass of picophytoplankton was 1 gm(-2), and the respective contributions of each group to the biomass were 69, 3 and 28%. In the HNLC waters, Prochlorococcus cells were slightly less numerous than in the warm pool, whereas the other groups were several times more abundant (from 3 to 5 times). Abundance of Prochlorococcus, Synechococcus and picoeukaryotes were 1.2 x 10(13), 6.2 x 10(11) and 5.1 x 10(11) cells m(-2), respectively. The integrated biomass was 1.9 g C m(-2). Prochlorococcus was again the dominant group in terms of abundance and biomass (chlorophyll, carbon) ; the respective contributions of each group to the carbon biomass were 58, 7 and 35%. In the warm pool the total chlorophyll biomass was 28 mg m(-2), 57% of which was divinyl chlorophyll a. In the HNLC waters, the total chlorophyll biomass was 38 mg m(-2), 44% of which was divinyl chlorophyll a. Estimates of Prochlorococcus, Synechococcus and picoeukaryotes cell size were made in both hydrological conditions. (C) 2000 Elsevier Science Ltd. All rights reserved.

Boddy L, Wilkins MF, Morris CW (2001) Pattern recognition in flow cytometry. Cytometry 44 :195-209


BACKGROUND : Analytical flow cytometry (AFC), by quantifying sometimes more than 10 optical parameters on cells at rates of approximately 10(3) cells/s, rapidly generates vast quantities of multidimensional data, which provides a considerable challenge for data analysis. We review the application of multivariate data analysis and pattern recognition techniques to flow cytometry. METHODS : Approaches were divided into two broad types depending on whether the aim was identification or clustering. Multivariate statistical approaches, supervised artificial neural networks (ANNs), problems of overlapping character distributions, unbounded data sets, missing parameters, scaling up, and estimating proportions of different types of cells comprised the first category. Classic clustering methods, fuzzy clustering, and unsupervised ANNs comprised the second category.We demonstrate the state of the art by using AFC data on marine phytoplankton populations. RESULTS AND CONCLUSIONS : Information held within the large quantities of data generated by AFC was tractable using ANNs, but for field studies the problem of obtaining suitable training data needs to be resolved, and coping with an almost infinite number of cell categories needs further research.

Bouvier T, Troussellier M, Anzil A, Courties C, Servais P (2001) Using light scatter signal to estimate bacterial biovolume by flow cytometry. Cytometry 44 :188-194


BACKGROUND : In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS : The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS : With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS : Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

Brussaard CPD, Marie D, Thyrhaug R, Bratbak G (2001) Flow cytometric analysis of phytoplankton viability following viral infection. Aquatic Microbial Ecology 26 :157-166

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Two flow cytometric assays using physiological probes were used on the phytoplankton species Phaeocystis pouchetii and Micromonas pusilla to examine the assays’ utility in detecting viral infections. Dead cells were detected using the membrane impermeant nucleic-acid dye SYTOX-Green, which stains algal cells that have lost their membrane integrity. Live cells were detected using the membrane permeant dye Calcein-AM, which is hydrolyzed by intracellular esterases into a green fluorescent charged form, We found that both assays are easy to use, are reproducible and can indeed be used as markers of the viability of individual phytoplankton cells following infection by viruses. Cell death rates up to 0.8 d(-1) for P. pouchetii and 0.5 d(-1) for Al. pusilla were calculated. The first day postinfection, death rates determined by the Calcein-AM assay were typically twice as high as those determined by the SYTOX-Green assay. Both viability tests were found to assess the physiological status of noninfected P. pouchetii cells, independent of viral infection. The optimal choice of viability assay depended on the phytoplankton species studied. Compared with existing assays, the protocols described permit examination of infected phytoplankton in more detail, yielding insight into the heterogeneity of the algal population.

Button DK, Robertson BR (2001) Determination of DNA content of aquatic bacteria by flow cytometry. Appl Environ Microbiol 67 :1636-1645


The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4’,6’-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells ("dims"). The dims could be formed from laboratory cultivars ; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell(-1) was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell(-1).

Button DK, Robertson BR, Quang P (2001) Isolation of oligobacteria. Methods in Microbiology, Vol 30 30 :161-173

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Casotti R, Brunet C, Graziano A (2001) Ultraplankton of the Gulf of Naples by flow cytometry. Mediterranean Ecosystems : Structures and Processes :171-180

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Ultraplankton composition and distribution were investigated as a function of physical structures and water masses of the Gulf of Naples in November 1995. Cyanobacteria were distributed all over the Gulf but mostly in the surface mixed layer. Prochlorophytes were more abundant offshore and at depth. Small eukaryotes were typical of the surface of coastal stations. Prochlorophytes vertical distribution showed a peculiar profile, with two populations at depth, where the Modified Atlantic Water (MAW) was present. A periodic sampling of a fixed station in the Gulf of Naples started in June 1998 in order further to investigate environmental parameters ru ling this vertical distribution. Preliminary data interpretation is discussed. During the November cruise, bacterial abundances were correlated to chlorophyll a concentrations, and, despite the strong oligotrophy of the area, bacterial carbon never exceeded phytoplankton carbon.

Castberg T, Larsen A, Sandaa RA, Brussaard CPD, Egge JK, Heldal M, Thyrhaug R, van Hannen EJ, Bratbak G (2001) Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta). Marine Ecology-Progress Series 221 :39-46

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Several previous studies have shown that Emiliania huxleyi blooms and terminations have been succeeded by an increase in large virus-like particles (LVLP), strongly suggesting the bloom collapse was caused by viral lysis. However, due to methodological limitations, knowledge of how such blooms affect the rest of the microbial community is limited. In the current study we induced a bloom of E. huxleyi in seawater enclosures and applied methods enabling us to describe the algae, bacteria and virus communities with greater resolution than has been done previously, The development of the dominating algal, viral and bacterial populations in the nutrient-amended seawater enclosures was followed by flow cytometry (FCM). Light microscopy (LM), PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) were used to describe the changes in community composition in greater detail. The algal community was dominated by E. huxleyi until termination of the bloom by viral lysis, After bloom termination the additional algal populations present in the enclosures increased in abundance. A marked increase in viruses other than the one infecting E. huxleyi was also observed. Total bacterial number and community composition were also greatly influenced by the bloom and its collapse.

Cavender-Bares KK, Karl DM, Chisholm SW (2001) Nutrient gradients in the western North Atlantic Ocean : Relationship to microbial community structure and comparison to patterns in the Pacific Ocean. Deep-Sea Research Part I-Oceanographic Research Papers 48 :2373-2395

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Studies of nitrogen and phosphorus dynamics in the oligotrophic surface waters of the western North Atlantic Ocean have been constrained because ambient concentrations are typically at or below the detection limits of standard colorometric methods, except during periods of deep vertical mixing. Here we report the application of high-sensitivity analytical methods-determinations of nitrate plus nitrite (N + N) by chemiluminescence and soluble reactive phosphorus (SR-P) by the magnesium induced co-precipitation (MAGIC) protocol-to surface waters along a transect from the Sargasso Sea at 26 degreesN through the Gulf Stream at 37 degreesN, including sampling at the JGOFS Bermuda Atlantic Time-series Study (BATS) station. The results were compared with data from the BATS program, and the HOT station in the Pacific Ocean, permitting cross-ecosystem comparisons. Microbial populations were analyzed along the transect, and an attempt was made to interpret their distributions in the context of the measured nutrient concentrations.
Surface concentrations of N+N and SRP during the March 1998 transect separated into 3 distinct regions, with the boundaries corresponding roughly to the locations of the BATS station (similar to 31 degreesN) and the Gulf Stream (similar to 37 degreesN). Although N+N and SRP co-varied, the [N+N] :[SR-P] molar ratios increased systematically from similar to1 to 10 in the southern segment, remained relatively constant at similar to 40-50 between 31 degreesN and 37 degreesN, then decreased again systematically to ratios < 10 north of the Gulf Stream. Dissolved organic N (DON) and P (DOP) dominated (greater than or equal to 90%) the total dissolved N (TDN) and P (TDP) pools except in the northern portion of the transect. The [DON] : [DOP] molar ratios were relatively invariant (similar to 30-60) across the entire transect.
Heterotrophic prokaryotes (operationally defined as "bacteria"), Prochlorococcus, Synechococcus, ultra-and nanophytoplaakton, cryptophytes, and coccolithophores were enumerated by flow cytometry. The abundance of bacteria was well correlated with the concentration of SRP, and that of the ultra- and nanophytoplankton was well correlated with the concentration of N+N. The only group whose concentration was correlated with temperature was Prochlorococcus, and its abundance was unrelated to the concentrations of nutrients measured at the surface.
We combined our transect results with time-series measurements from the BATS site and data from select depth profiles, and contrasted these North Atlantic data sets with time-series of N and P nutrient measurements from a station in the North Pacific subtropical gyre near Hawaii [Hawaii Ocean Time-series (HOT) site]. Two prominent differences are readily observed from this comparison. The [N + N] : [SRP] molar ratios are much less than 16 : 1 during stratified periods in surface waters at the BATS site, as is the case at the HOT site year round. However, following deep winter mixing, this ratio is much higher than 16, 1 at BATS. Also, SRP concentrations in the upper 100 m at BATS fall in the range 1-10 nM during stratified periods, which is at least one order of magnitude lower than at the HOT site. That two ecosystems with comparable rates of primary and export production would differ so dramatically in their nutrient dynamics is intriguing, and highlights the need for detailed cross ecosystem comparisons. (C) 2001 Elsevier Science Ltd. All rights reserved.

Chase JC, Dawson-Coates JA, Haddow JD, Stewart MH, Haines LR, Whitaker DJ, Kent ML, Olafson RW, Pearson TW (2001) Analysis of Kudoa thyrsites (Myxozoa : Myxosporea) spore antigens using monoclonal antibodies. Diseases of Aquatic Organisms 45 :121-129

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A method employing Percoll (TM) gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K, thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K, thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K, thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.

Chen F, Lu JR, Binder BJ, Liu YC, Hodson RE (2001) Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold. Appl Environ Microbiol 67 :539-545


A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

Chiu LC, Ooi VE, Wan JM (2001) Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 19 :845-849


Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (20:5omega3) found primarily in aquatic organisms. We have shown previously that EPA inhibits the growth but is not toxic to human leukemic K-562 cells. In this study, the anti-proliferative effect of EPA on the leukemic cells was further determined and its impacts on cell cycle progression and cyclin expression were investigated. EPA inhibited proliferation of K-562 cells, which was associated with accumulation of G0/G1 cells and down-regulation of cyclin E expression. Cyclin B1-expressing cells were also reduced showing that down-regulation of cyclin expression might be important in the anti-proliferation of EPA.

Christaki U, Giannakourou A, Van Wambeke F, Gregori G (2001) Nanoflagellate predation on auto- and heterotrophic picoplankton in the oligotrophic Mediterranean Sea. Journal of Plankton Research 23 :1297-1310

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Dynamics of autotrophic and heterotrophic prokaryotes and their consumption by nanoflagellates were studied in the euphotic zone at nine stations located from the Levantine Basin (34 degreesE) to the Balearic sea (5 degreesE) in June 1999. Bacterial biomass constituted the largest proportion of living biomass at all stations. Integrated bacterial production at the furthest east station, was sixfold lower than integrated bacterial production at the furthest west (13 and 75 mg C m(-2) d(-1) respectively). Estimated heterotrophic nanoflagellate bacterivory accounted for 45-87% of bacterial production. Small protists (<3 m) dominated the bacterivore assemblage and accounted for more than 90% of the heterotrophic bacterial consumption. Our results indicated that there was no negative selection against Synechococcus and that both picoplankton groups were grazed according to their standing stocks. An estimated consumption of Synechococcus derived from food vacuole content analysis of nanoflagellates revealed that they consumed from 0.5 to 45% (mean 13%) of Synechococcus stock per day. These data are among the first documenting the relative grazing impact of heterotrophic nanoflagellates on bacteria and Synechococcus in situ. Assuming that there was no selection for or against Prochlorococcus, heterotrophic nanoflagellates could ingest from 1.4 to 21% (mean 6%) of Prochlorococcus stock per day. The amount of organic carbon obtained by heterotrophic nanoflagellates from photosynthetic prokaryotes represented 27% of the total amount of carbon obtained from total prokaryotes.

Cleven EJ, Weisse T (2001) Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance. Aquatic Microbial Ecology 23 :147-161

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We investigated the taxonomic composition of the heterotrophic nanoflagellate (HNF) assemblage and its taxon-specific bacterial grazing rates in Lake Constance (Germany) over the course of 1 yr. Bacterial grazing rates were measured using natural fluorescently labelled bacteria (FLB) and compared to bacterial production estimated by the uptake of C-14-leucine incorporation. Glutaraldehyde-fixed, DAPI-stained flagellates were counted using epifluorescence microscopy. Based on annual averages, small species such as Spumella sp. (2 to 6 mum) were the most numerous HNF and the dominant bacterivores. Larger flagellates such as Kathablepharis sp, contributed significantly to total HNF biomass, in particular during spring, but were relatively unimportant as bacterial grazers. The HNF community structure changed during the transition from the phytoplankton spring bloom to the clearwater phase, with small flagellates such as heterokonts, kinetoplastids and choanoflagellates becoming increasingly abundant. The flagellate community composition was more diverse during summer and autumn than in spring. Per capita ingestion rates ranged from 0 to 31 bacteria HNF-1 h(-1) and changed seasonally up to 10-fold within a given taxon. Mixotrophic species contributed little to total bacterivory. We provide evidence that the relative significance of bacterial ingestion by a given flagellate taxon may change seasonally. Based upon our experimental results, we discuss potential shortcomings inherent in the FLB technique.

Cotner JB, Ogdahl ML, Biddanda BA (2001) Double-stranded DNA measurement in lakes with the fluorescent stain PicoGreen and the application to bacterial bioassays. Aquatic Microbial Ecology 25 :65-74

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We used the double-stranded DNA (dsDNA) stain PicoGreen with a microplate fluorometer to measure bacterial abundance, biomass, and growth rates in lake water. PicoGreen fluorescence units (PFU) correlated closely with bacterial abundance measured with acridine orange direct counts (R-2 = 0.95 to 0.98) as well as bacterial biomass inferred from image analysis (R-2 = 0.95 to 0.98) in eutrophic waters. PicoGreen fluorescence increased proportionally with bacterial size, indicating that it was a good indicator of biomass as well as abundance. In oligotrophic Lake Superior, there was a weaker, but significant (p < 0.05) correlation between PFU and abundance (R-2 = 0.52) as well as PFU and biomass (R-2 = 0.54). Growth rate measurements in bottle cultures showed a similar relationship, with PFU, abundance, and biomass being more tightly correlated in productive waters than in oligotrophic waters. Parallel dilution cultures were performed in microplate wells (nanocosms) and 11 bottles, The slope of nanocosm fluorescence to bottle fluorescence was ca 1, indicating that nanocosms mimicked bacterial abundance and growth in bottle cultures. Preservation of Escherichia coli with formaldehyde showed that there was an initial loss of dsDNA of about 10 to 15% with little subsequent loss for 2 wk, indicating that bacteria from growth experiments conducted in the field could be preserved for subsequent analysis in the laboratory, Bacterial cellular dsDNA content in 3 Minnesota lakes varied between 0.6 and 6.2 fg cell(-1) and was highest in the most eutrophic and most rapidly growing bacterial community, i.e., the more eutrophic lakes. These results suggest that the PicoGreen method is effective for growth bioassays in systems with moderate to high levels of bacterial productivity. PicoGreen coupled with a microplate fluorescence reader is a promising method for determining bacterial biomass and growth rates, especially in meso- to eutrophic systems.

Courties C, Troussellier M (2001) Introduction - Flow cytometry in the marine environment. Cytometry 44 :163-163

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Cucci TL, Sieracki ME (2001) Effects of mismatched refractive indices in aquatic flow cytometry. Cytometry 44 :173-178


BACKGROUND : Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural waters. The refractive index of water samples from aquatic environments can differ because of a variety of factors such as dissolved organic content, aldehyde preservative, sample salinity, and temperature. In flow cytometric analyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We measured the effect of this mismatch on cell size measurements. METHODS : We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity ratio. The effects were characterized for standard microspheres, cultured phytoplankton cells of different sizes, and natural populations from an estuarine river. RESULTS : We found that the distorted scatter signals resulted in an increase in the apparent size of small cells (1—2 microm) by a factor of 4.5 times. Cells in the size range of 3—5 microm were less affected by the salinity differences, and cells larger than 5 microm were not affected. Chlorophyll and phycoerythrin fluorescences and 90 degrees light scatter signals were not changed by sheath and sample salinity differences. CONCLUSIONS : Care must be taken to ensure that the sheath and sample refractive index are matched when using forward light scatter to measure cell size spectra, especially in estuarine studies, where salinity can vary greatly. Of the factors considered that can change the sample refractive index, salinity gradients in an estuary cause the largest index mismatch and, consequently, the largest error in scatter.

Diez B, Pedros-Alio C, Marsh TL, Massana R (2001) Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques. Applied and Environmental Microbiology 67 :2942-2951

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We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea, Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked, The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample, The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions ; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses, DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes, In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

Diez B, Pedros-Alio C, Massana R (2001) Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing. Applied and Environmental Microbiology 67 :2932-2941

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Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system, Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters, Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes, Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes. especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi, There were tyro relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

Ducklow HW, Smith DC, Campbell L, Landry MR, Quinby HL, Steward GF, Azam F (2001) Heterotrophic bacterioplankton in the Arabian Sea : Basinwide response to year-round high primary productivity. Deep-Sea Research Part Ii-Topical Studies in Oceanography 48 :1303-1323

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Heterotrophic bacterial abundance and productivity were measured during five and four cruises, respectively, in the northwest Arabian Sea as part of the US JGOFS Process Study, which provided a new view of seasonal bacterial dynamics in that part of the basin influenced by monsoonal forcing. In this paper, surface layer data are used to address two questions concerning the influence of the monsoon cycle on bacterial dynamics : (1) Is there a bacterial bloom in the SW Monsoon ? and (2) Is bacterial production low during the oligotrophic Spring Intermonsoon ? An extensive comparison of epifluorescence microscopy and flow cytometry, unprecedented at this scale, detected essentially the same heterotrophic bacterial populations and distributions, with some between-cruise differences. Use of the two methods allowed us to extend our observations in space and time. Bacterial productivity, both in the surface layer and integrated over the euphotic zone, was elevated less than 2-fold during the Southwest Monsoon. Levels of bacterial abundance and production were low during the Northeast Monsoon, then increased in March during the Spring Intermonsoon. There was some stimulation of abundance or production inshore in response to coastal upwelling. In general, the basin was enriched in bacterial biomass > 5 x 10(8) cells 1(-1) throughout the year, relative to other tropical regimes, presumably in response to overall high PP and DOC levels. Seasonally uniform DOC levels may be regulated in part by intense bacterial utilization rates, but also reflect seasonal consistency in PP. (C) 2001 Elsevier Science Ltd. All rights reserved.

Dusenberry JA, Olson RJ, Chisholm SW (2001) Photoacclimation kinetics of single-cell fluorescence in laboratory and field populations of Prochlorococcus. Deep-Sea Research Part I-Oceanographic Research Papers 48 :1443-1458

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Picophytoplankton show promise as dynamic tracers for vertical mixing if an appropriate index of photoacclimative state and the kinetics of change in that index can be determined. To this end, several light shift incubations were carried out with laboratory cultures of Prochlorococcus MED4 and natural populations of Prochlorococcus at sea. These time series revealed systematic changes in chlorophyll fluorescence in response to changes in incident light intensity. Prochlorococcus also exhibited strong diel patterns in fluorescence and light scattering resulting from the phasing of cell division to the daily photoperiod, In order to obtain a parameter that was insensitive to the daily photoperiod, Prochlorococcus red fluorescence was normalized to the 0.33 power of forward angle light scattering, an empirically derived normalization factor. We propose that this normalized fluorescence could be an appropriate measure of photoacclimation in Prochlorococcus, suitable for use in inverse modeling of mixing dynamics. A logistic model for photoacclimation was found to fit the experimental data well, although there was some systematic deviation from the data for populations shifted to high irradiances. Estimates of photoacclimative rates ranged from 0.9 to 2 d(-1). (C) 2001 Elsevier Science Ltd. All rights reserved.

Eguchi M, Ostrowski M, Fegatella F, Bowman J, Nichols D, Nishino T, Cavicchioli R (2001) Sphingomonas alaskensis strain AFO1, an abundant oligotrophic ultramicrobacterium from the North Pacific. Applied and Environmental Microbiology 67 :4945-4954

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Numerous studies have established the importance of picoplankton (microorganisms of less than or equal to2 mum in length) in energy flow and nutrient cycling in marine oligotrophic environments, and significant effort has been directed at identifying and isolating heterotrophic picoplankton from the world’s oceans. Using a method of diluting natural seawater to extinction followed by monthly subculturing for 12 months, a bacterium was isolated that was able to form colonies on solid medium. The strain was isolated from a 10(5) dilution of seawater where the standing bacterial count was 3.1 X 10(5) cells ml(-1). This indicated that the isolate was representative of the most abundant bacteria at the sampling site, 1.5 km from Cape Muroto, Japan. The bacterium was characterized and found to be ultramicrosized (less than 0.1 mum(3)), and the size varied to only a small degree when the cells were starved or grown in rich media. A detailed molecular (16S rRNA sequence, DNA-DNA hybridization, G+C mol%, genome size), chemotaxonomic (lipid analysis, morphology), and physiological (resistance to hydrogen peroxide, heat, and ethanol) characterization of the bacterium revealed that it was a strain of Sphingomonas alaskensis. The type strain, RB2256, was previously isolated from Resurrection Bay, Alaska, and similar isolates have been obtained from the North Sea. The isolation of this species over an extended period, its high abundance at the time of sampling, and its geographical distribution indicate that it has the capacity to proliferate in ocean waters and is therefore likely to be an important contributor in terms of biomass and nutrient cycling in marine environments.

Eschbach E, Reckermann M, John U, Medlin LK (2001) A simple and highly efficient fixation method for Chrysochromulina polylepis (Prymnesiophytes) for analytical flow cytometry. Cytometry 44 :126-132


BACKGROUND : To study the fragile Prymnesiophyte species Chrysochromulina polylepis by flow cytometry (FC), we needed an effective fixation method. This method must guarantee a high yield of fixed cells to achieve acceptable measurement times by FC and to allow quick processing of many samples. Moreover, we wanted a method that allows for storage of fixed samples when FC analysis cannot be done immediately. METHODS : Different aldehydes and methanol were tested at different final concentrations. Gravity sedimentation and centrifugation were applied to achieve higher cell concentrations. Storage of fixed samples was tested under different conditions. RESULTS : 0.25% glutaraldehyde (GA) fixation yielded a recovery rate of about 90%. The signals obtained by FC analysis were excellent. It is possible to centrifuge GA-fixed cells and to store them for several weeks. CONCLUSIONS : GA is the fixative of choice for FC analysis of C. polylepis (and possibly other small delicate species) because it yielded highly significant recovery rates and high-quality FC signals. Cells can be centrifuged to increase the cell concentration, thereby achieving short measurement times with FC. The possibility of long-term storage of fixed cells presents an additional advantage if FC analysis cannot be done immediately.

Fouilland E, Courties C, Descolas-Gros C (2001) Size-fractionated carboxylase activities during a 32 h cycle at 30 m depth in the north-western Mediterranean Sea after an episodic wind event. Journal of Plankton Research 23 :623-632

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Using size fractionation filtration (1 mum), we associated carboxylase activities (Rubisco, beta -carboxylases) and chlorophyll measurements with cell enumeration by flow cytometry at a permanent site of the central Ligurian,Sea in the north-western Mediterranean Sea (73 degrees 25 ’N-7 degrees 51 ’E). The analyses were carried out over a day/night Cycle (at 30 m depth) following a strong wind event, during the transition period from spring mesotrophic to summer oligotrophic conditions. The highest values of Rubisco activity and beta -carboxylase activity per chlorophyll a (Chl a) for >1 mum cells were observed during the light period of the cycle, reaching 18.9 and 4.3 nmol CO2 (mug Chl a)(-1) h(-1), respectively. This higher activity is assumed to be correlated with a dominance of nanoflagellates in the phytoplankton community. Such phytoplankton species generally had higher beta -carboxylase activity, expressed as a percentage of Rubisco activity (the betaC/R ratio), than diatoms. Using ooze, cytometry analysis to enumerate those cells <1 mum in size, we followed the values of Rubisco activity and pigment content expressed per cell, for picophytoplankton cells. The photoautotrophic activity, measured as the in vitro Rubisco activity for small picoeukaryote cells, was higher than for cyanobacteria cells with lower apparent cell size. These results suggested an optimum of CO2 assimilation reached by the pico- and nano phytoplankton in accordance with the cell size and growth rates from previous observations in the literature.

Fournier M, Pellerin J, Clermont Y, Morin Y, Brousseau P (2001) Effects of in vivo exposure of Mya arenaria to organic and inorganic mercury on phagocytic activity of hemocytes. Toxicology 161 :201-211


Marine bivalves are aquatic invertebrate organisms which can be used as bioindicators in environmental monitoring. In vivo effects of mercuric chloride (HgCl(2)) and methylmercury (CH(3)HgCl) on phagocytic function of Mya arenaria hemocytes were evaluated in this study. Clams were exposed to single metal in water for up to 28 days at concentrations ranging from 10(-9) to 10(-5) M. Phagocytic activity of hemocytes was determined by uptake of fluorescent microspheres and flow cytometry. All clams exposed to 10(-5) M HgCl(2) died by day 7 of exposure. The viability of hemocytes was decreased only in clams exposed to 10(-6) M HgCl(2) for 28 days. A significant decrease in phagocytic activity of hemocytes was observed in clams exposed to 10(-6) M of HgCl(2) for 28 days. A similar pattern was observed with CH(3)HgCl, but at an earlier time. Chemical analysis performed on the tissues of the animals clearly show a greater uptake of the organic form of mercury by clams. Furthermore, a clear correlation was established between body burden of mercury and effects on phagocytic activity of hemocytes. Overall, the results of this study show that both speciations of mercury inhibited phagocytic function of Mya arenaria hemocytes following in vivo exposures.

Franklin NM, Adams MS, Stauber JL, Lim RP (2001) Development of an improved rapid enzyme inhibition bioassay with marine and freshwater microalgae using flow cytometry. Arch Environ Contam Toxicol 40 :469-480


A rapid toxicity test based on inhibition of esterase activity in marine and freshwater microalgae (Selenastrum capricornutum, Chlorella sp., Dunaliella tertiolecta, Phaeodactylum tricornutum, Tetraselmis sp., Entomoneis cf. punctulata, Nitzschia cf. paleacea) was developed using flow cytometry. Uptake of fluorescein diacetate (FDA) was optimized for each species by varying the substrate concentration, incubation time, and media pH. Propidium iodide (PI) was utilized to assess membrane integrity. The optimized FDA/PI staining procedure was then used to assess the toxicity of copper in short-term exposures (1-24 h). Esterase activity was a sensitive indicator of copper toxicity in S. capricornutum and E. cf. punctulata. As copper concentrations increased, esterase activity decreased in a concentration-dependent manner. The 3- and 24-h EC50 values (based on mean activity states) were 112 microg Cu L(-1) (95% confidence limits 88-143) and 51 microg Cu L(-1) (95% confidence limits 38-70) for S. capricornutum and 47 microg Cu L(-1) (95% confidence limits 43-51) and 9.1 microg Cu L(-1) (95% confidence limits 7.6-11) for E. cf. punctulata, respectively. This enzyme inhibition endpoint showed similar sensitivity to chronic growth rate inhibition in E. cf. punctulata (48-h and 72-h EC50 values of 17 and 18 microg L(-1), respectively) but was less sensitive compared to growth for S. capricornutum (48-h and 72-h EC50 values of 4.9 and 7.5 microg L(-1), respectively). For the other five species tested, inhibition of FDA fluorescence was relatively insensitive to copper, even at copper concentrations that severely inhibited cell division rate. These short-term bioassays that detect sublethal endpoints may provide a more rapid and cost-effective way of monitoring contaminant impacts in natural waters.

Franklin NM, Stauber JL, Lim RP (2001) Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters. Environ Toxicol Chem 20 :160-170


Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry : cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.

Fuchs BM, Syutsubo K, Ludwig W, Amann R (2001) In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 67 :961-968


One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973-4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083(T) was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows : class I, 3% ; class II, 21% ; class III, 35% ; class IV, 18% ; class V, 16% ; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.

Fujiwara H, Matsunaga K, Saito M, Hagiya S, Furukawa K, Nakamura H, Ohizumi Y (2001) Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells. Eur J Pharmacol 413 :37-45


In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tet rone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC(50) value ; 10 microM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 microM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-di methyl-[1S-(1 alpha,6b alpha,9a beta,11 alpha,11b beta)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 microM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC(50) value ; 10 microM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12 bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC(50) value ; 3 microM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.

Garcia MT, Ribosa I, Guindulain T, Sanchez-Leal J, Vives-Rego J (2001) Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment. Environ Pollut 111 :169-175


The effect of the alkyl chain of quaternary ammonium-based surfactants on their aquatic toxicity and aerobic biodegradability has been studied. Two families of monoalkylquats surfactants were selected : alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium halides. Acute toxicity tests on Daphnia magna and Photobacterium phosphoreum were carried out and EC50 values in the range of 0.1-1 mg/l were obtained for the two series of cationic surfactants. Although the substitution of a benzyl group for a methyl group increases the toxicity, an incremental difference in toxicity between homologs of different chain length were not observed. Biodegradability of the different homologs was determined not only in standard conditions but also in coastal water, both tests yielding similar results. An increase in the alkyl chain length or the substitution of a benzyl group for a methyl group reduces the biodegradation rate. The degradation of these compounds in coastal waters was associated with an increase in bacterioplankton density, suggesting that the degradation takes place because the compound is used as a growth substrate.

Garcia-Abiado MA, Rinchard J, Dabrowski K (2001) Meiotic gynogenesis induction in muskellunge Esox masquinongy by thermal and pressure shocks. Journal of the World Aquaculture Society 32 :195-201

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Mass production of fast-growing, all-female muskellunge Esox masquinongy by gynogenesis requires optimized techniques of preventing second polar body extrusion. Heat, cold, and pressure shocks were evaluated for their efficiency of doubling the maternal genome. Muskellunge eggs (20-40 g) were activated with 1 mL ultraviolet (UV)-irradiated (1,248 J/ m(2)) heterologous sperm of yellow perch Perca flavescens. Survival and ploidy (by flow cytometry) were determined during the eyed-stage. Cold shocks of 1.3 +/- 1 C were applied at 5 or 20 min after gamete activation with water (time of initiation, TI) for a duration of 150 min and pressure shocks of 48,263 or 55,158 kPa (7,000 or 8,000 psi, respectively) at a TI of 4 min for 12 min. These shock treatments resulted in 43.7-95.0% diploid gynogens with corresponding yield of diploid gynogen (percent diploid gynogens X total percent survival) of 2.6-11.1%. Cold shocks applied at TI of 5 or 20 min after activation resulted in statistically similar percent survival, percent diploid gynogens, and yield of diploid gynogens. Heat shocks of 31 +/- 0.1 C applied at a TI of 5 to 15 min for a duration of 5 min resulted in 4.8-21.1% diploid gynogens with yields of 0.1-0.4%. Cold and pressure shocks have better potential than heat shock for preventing the second polar body extrusion. Muskellunge eggs activated with UV-irradiated yellow perch sperm, but not exposed to shock, resulted in 100% haploids with survival of 2.3-5.8%. No viable embryos were produced from the hybrid cross between muskellunge and yellow perch, thus, all diploids produced after the shock treatments were unambiguous meiotic gynogens. Muskellunge eggs fertilized with fresh muskellunge sperm (controls) showed 60.4-64.0% survival to the eyed-stage and 100% diploidy. Considering that the sex-determining mechanism in muskellunge follows the WZ female, ZZ male system, future efforts should be directed to test the efficiency of cold and pressure shocks for mass-producing gynogenetic super female (WW) muskellunge.

Garczarek L, Partensky F, Irlbacher H, Holtzendorff J, Babin M, Mary I, Thomas JC, Hess WR (2001) Differential expression of antenna and core genes in Prochlorococcus PCC 9511 (Oxyphotobacteria) grown under a modulated light-dark cycle. Environ Microbiol 3 :168-175


The continuous changes in incident solar light occurring during the day oblige oxyphototrophs, such as the marine prokaryote Prochlorococcus, to modulate the synthesis and degradation rates of their photosynthetic components finely. How this natural phenomenon influences the diel expression of photosynthetic genes has never been studied in this ecologically important oxyphotobacterium. Here, the high light-adapted strain Prochlorococcus sp. PCC 9511 was grown in large-volume continuous culture under a modulated 12 h-12 h light-dark cycle mimicking the conditions found in the upper layer of equatorial oceans. The pcbA gene encoding the major light-harvesting complex showed strong diel variations in transcript levels with two maxima, one before the onset of illumination and the other near the end of the photoperiod. In contrast, the mRNA level of psbA (encoding the reaction centre II subunit D1), the monocistronic transcript of psbD (encoding D2) and the dicistronic transcript of psbDC were all tightly correlated with light irradiance, with a minimum at night and a maximum at noon. The occurrence of a second peak during the dark period for the monocistronic transcript of psbC (encoding one of the PS II core Chl a antenna proteins) suggested the involvement of post-transcriptional regulation. Differential expression of the external antenna and core genes may constitute a mechanism of regulation of the antenna size to cope with the excess photon fluxes that Prochlorococcus cells experience in the upper layer of oceans around midday. The 5’ ends of all transcripts were mapped, and a conserved motif, 5’-TTGATGA-3’, was identified within the putative psbA and pcbA promoters.

Gomez M, Mayo I, Torres S (2001) Flow cytometry of cell proliferation through the incorporation of bromodeoxyuridine as an index of growth rate in the water flea, Daphnia magna (Crustacea, Cladocera). Cytometry 44 :264-271

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Background : In this paper, we used a small crustacean as a model to develop a method for quantifying growth rates through the measurement of a cell proliferation marker. This was done in order to study the feasibility of this assay for estimating zooplankton production in the ocean. Flow cytometry immunodetection of bromodeoxyuridine (BrdU) was performed to detect and quantify the cycling nuclei of Daphnia magna.
Methods : A combination of mechanical dissociation and cell enucleation procedures proved to be the most convenient method for preparing nuclear suspensions from whole organisms. Up to three populations of nuclei with different ploidy were observed. The relative abundance of these nuclear populations changed with the size of the nea.
Results : The staining technique has been optimized. The time and concentration for the maximum detection of BrdU-labeled nuclei were 3 h at 300 muM BrdU. Whole organisms can be frozen (-20 degreesC) after incubation with no changes in the final results. The method was used in different physiological conditions under controlled food and temperature in order to test the inverse relationship between physiological rates and size of organisms at several developmental stages. The quantification of BrdU-labeled nuclei in 1-6 day-old larvae showed the highest labeling index, with a mean of 95 +/- 1% (n = 22). In contrast, young animals (0.8-1.2 mm) had 25 +/- 4% (n = 16, P < 0.001) and adults (>1.4mm) had 14 +/- 3% (n = 4, P < 0.001). The results obtained show an expected tendency, suggesting that a direct relationship exists between the labeling index and the instantaneous growth rate.
Conclusions : Certain features of our method, such as the short times required for labeling and the possibility of preserving the samples during field experiments and under different conditions (including natural concentrations and types of food), are advantageous to the study of processes governing energy fluxes in pelagic ecosystems. (C) 2001 Wiley-Liss, Inc.

Gregori G, Citterio S, Ghiani A, Labra M, Sgorbati S, Brown S, Denis M (2001) Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl Environ Microbiol 67 :4662-4670


The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green ; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Gregori G, Colosimo A, Denis M (2001) Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry. Cytometry 44 :247-256


BACKGROUND : The Bay of Marseilles is under the influence of a large urban concentration and its maritime activities. All of them discharge compounds (hydrocarbons, excess nutrients, heavy metals, chemicals, etc.) that can alter the marine ecosystem. To investigate whether ultraphytoplankton (<10 microm) could be used as biosensors for their own ecosystem, a 2-year survey was conducted in the Bay of Marseilles. METHODS : Seven stations monitored water mass and potential anthropic effects in the bay. Seawater samples were collected monthly or bimonthly at three depths, prefiltered, fixed, and kept in liquid nitrogen until flow cytometric analysis. RESULTS : Five categories were created : Prochlorococcus, picoeukaryotes (<2 microm), nanoeukaryotes I (2—6 microm), nanoeukaryotes II (6—10 microm), and Synechococcus (<1.5 microm). Artificial neural network analysis (Kohonen self-organizing maps) produced the same number of clusters as cluster analysis with Winlist software (Verity Software House). CONCLUSIONS : In addition to the wide variabilities in abundance and biomass, there were a strong seasonal signal and sporadic events. Lessons are derived from this study for future monitoring of marine microorganisms.

Hampel M, Moreno-Garrido I, Sobrino C, Lubian LM, Blasco J (2001) Acute toxicity of LAS homologues in marine microalgae : esterase activity and inhibition growth as endpoints of toxicity. Ecotoxicol Environ Saf 48 :287-292


The toxicity of two linear alkylbenzene sulfonate (LAS) homologues (C(10) and C(13)) was evaluated in four marine microalgae (Nannochloropsis gaditana, Tetraselmis suecica, Rhodomonas salina, and Isocrysis galbana), using growth inhibition rate and esterase activity (measured by flow cytometry) as endpoints. The inhibitor effect was higher for the C(13) LAS homologue than for C(11), in both responses analyzed. When both endpoints were compared, the growth inhibition rate was between 2 and 5 times more sensitive than esterase activity. Among microalgae species, R. salina exhibited the highest sensitivity.

Holtzendorff J, Partensky F, Jacquet S, Bruyant F, Marie D, Garczarek L, Mary I, Vaulot D, Hess WR (2001) Diel expression of cell cycle-related genes in synchronized cultures of Prochlorococcus sp. strain PCC 9511. J Bacteriol 183 :915-920


The cell cycle of the chlorophyll b-possessing marine cyanobacterium Prochlorococcus is highly synchronized under natural conditions. To understand the underlying molecular mechanisms we cloned and sequenced dnaA and ftsZ, two key cell cycle-associated genes, and studied their expression. An axenic culture of Prochlorococcus sp. strain PCC 9511 was grown in a turbidostat with a 12 h-12 h light-dark cycle for 2 weeks. During the light periods, a dynamic light regimen was used in order to simulate the natural conditions found in the upper layers of the world’s oceans. This treatment resulted in strong cell cycle synchronization that was monitored by flow cytometry. The steady-state mRNA levels of dnaA and ftsZ were monitored at 4-h intervals during four consecutive division cycles. Both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase. Western blot experiments indicated that the peak of FtsZ concentration occurred at night, i.e., at the time of cell division. Thus, the transcript accumulation of genes involved in replication and division is coordinated in Prochlorococcus sp. strain PCC 9511 and might be crucial for determining the timing of DNA replication and cell division.

Hood KA, West LM, Northcote PT, Berridge MV, Miller JH (2001) Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine. Apoptosis 6 :207-219


The marine sponge metabolites mycalamide A (mycalamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine-beta-D-arabinofuranoside (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.

Howard-Jones MH, Frischer ME, Verity PG (2001) Determining the physiological status of individual bacterial cells. Methods in Microbiology, Vol 30 30 :175-205

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Jacquet S, Partensky F, Lennon JF, Vaulot D (2001) Diel patterns of growth and division in marine picoplankton in culture. Journal of Phycology 37 :357-369

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The effect of a 12:12-h light:dark (LD) cycle on the phasing of several cell parameters was explored in a variety of marine picophytoplanktonic strains. These included the photosynthetic prokaryotes Pro-chlorococcus (strains MED 4, PCC 9511, and SS 120) and Synechococcus (strains ALMO 03, ROS 04, WH 7803, and WH 8103) and five picoeukaryotes (Bathycoccus prasinos Eikrem et Throndsen, Bolidomonas pacifica Guillou et Chretiennot-Dinet, Micromonas pusilla Manton et Parke, Pelagomonas calceolata Andersen et Saunders, and Pycnococcus provasolii Guillard et al.). Flow cytometric analysis was used to determine the relationship between cell light scatter, pigment fluorescence, DNA (when possible), and the LD cycle in these organisms. As expected, growth and division were tightly coupled to the LD cycle for all of these strains. For both Prochlorococcus and picoeukaryotes, chi and intracellular carbon increased throughout the light period as estimated by chi fluorescence and light scatter, respectively. In response to cell division, these parameters decreased regularly during the early part of the dark period, a decrease that either continued throughout the dark period or stopped for the second half of the dark period. For Synechococcus, the decrease of chi and scatter occurred earlier (in the middle of the light period), and for some strains these cellular parameters remained constant throughout the dark period. The timing of division was very similar for all picoeukaryotes and occurred just before the subjective dusk, whereas it was more variable between the different Prochlorococcus and Synechococcus strains. The burst of division for Prochlorococcus SS 120 and PCC 9511 was recorded at the subjective dusk, whereas the MED 4 strain divided later at night. Synechococcus ALMO 03, ROS 04, and WH 7803, which have a low phycourobilin to phycoerythrobilin (PUB:PEB) ratio, divided earlier, and their division was restricted to the light period. In contrast, the high PUB:PEB Synechococcus strain WH 8103 divided preferentially at night. There was a weak linear relationship between the FALS(max):FALS(min) ratio and growth rate calculated from cell counts (r = 0.83, n = 11, P < 0.05). Because of the significance of picoplanktonic populations in marine systems, these results should help to interpret diel variations in oceanic optical properties in regions where picoplankton dominates.

Jacquet S, Partensky F, Marie D, Casotti R, Vaulot D (2001) Cell cycle regulation by light in Prochlorococcus strains. Appl Environ Microbiol 67 :782-790


The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 micromol of quanta x m(-2) x s(-1), the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the "light on" signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.

Jiao NZ, Yang YH, Koshikawa H, Harada S, Watanabe M (2001) Microscopic overestimation of heterotrophic bacteria in open waters of China Seas. Journal of Microbiology and Biotechnology 11 :899-901

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Comparison of the abundances of heterotrophic bacteria in the East and South China Seas by standard epifluorescence microscopy and flow cytometry revealed that Prochlorococcus was miscounted as heterotrophic bacteria in DAPI stained samples. This could result in 5-31% overestimations of heterotrophic bacterial abundance in the study areas.

Jochem FJ (2001) Morphology and DNA content of bacterioplankton in the northern Gulf of Mexico : analysis by epifluorescence microscopy and flow cytometry. Aquatic Microbial Ecology 25 :179-194

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The distribution of pelagic bacteria was assessed along 2 offshore - onshore transects in the northwestern Gulf of Mexico in July and October 1999 and along a salinity gradient (0.2 to 34.4 parts per thousand) in the Mississippi River plume in May 2000. Cell abundance was estimated by epifluorescence microscopy after DAPI staining and by flow cytometry after DNA staining with SYBR Green I. Total bacterial counts by both techniques corresponded well. Bacterial abundance ranged from 0.9 x 10(6) to 1.35 x 10(6) cells ml(-1) in the upper 200 m of the water column in the northwestern Gulf and from 0. 1 x 10(6) to 2.05 x 10(6) cells ml(-1) in the Mississippi River plume. Bacteria exhibited surface maxima in July 1999 but subsurface maxima in the upper half of the chlorophyll maximum in October 1999 and off the Louisiana shelf break in May 2000, Stations with a thin layer of low-salinity plume water exhibited an additional bacterial maximum at the surface. Within the Mississippi River plume, bacterial abundance decreased with increasing salinity, and their maximum abundance preceded the chlorophyll maximum along the salinity gradient. Three morphotypes of bacteria were distinguished by epifluorescence microscopy : cocci, rod-shaped bacteria, and curved bacteria. Cocci (40 to 60 % of total bacteria ; counts corrected for Prochlorococcus spp.) were the most common morphotype, Rods and curved bacteria had similar shares (18 to 25%) and presented multi-species consortia as indicated by the variability in size and shape of cells within each group. Flow cytometry revealed 4 bacterial subpopulations distinguished by their DNA content, none of which seem to reflect a specific morphotype. Whereas regional differences in the contribution of the distinguished DNA types to total bacterial abundance were low in the open Gulf, a switch in predominance from low-DNA to high-DNA cells below the subsurface chlorophyll maximum was obvious in all profiles. The ecological significance of bacterial DNA types as revealed by flow cytometry is discussed in the context of published results.

John EH, Davidson K (2001) Prey selectivity and the influence of prey carbon:nitrogen ratio on microflagellate grazing. J Exp Mar Bio Ecol 260 :93-111


We investigated the influence of prey species and nutritional value, in terms of carbon:nitrogen (C:N) ratio, on prey selection by the predatory microflagellate Paraphysomonas vestita. Experiments were conducted with two phytoplankton prey species of similar diameter to remove size-specific grazing effects. Live cells of both low and high C:N ratio (ranging from 4.8 to 14 ; N-replete and N-deplete, respectively) were offered to the predator either individually or in combination. By utilising analytical flow cytometry, we were able to enumerate the two prey species and, hence, study selective predation in the mixed-prey assemblage. In single prey experiments, the maximum observed ingestion rates were found to be higher, at all prey C:N ratios, when Isochrysis galbana was the prey item when compared to Pavlova lutheri, whilst maximum specific predator division rates were similar for both prey. Ingestion rates were influenced by prey nutrient status, higher values being observed with N-replete than N-deplete prey. When the two prey species were presented to P. vestita as a mixture, I. galbana was ingested more rapidly than P. lutheri, although ingestion was found to be suppressed when compared to when this was the sole prey species. Conversely, the presence of I. galbana did not influence the rate of ingestion of P. lutheri. P. vestita was, therefore, able to modify its rate of ingestion on the basis of prey type and prey C:N ratio and to discriminate between alternative prey of similar size in mixed-prey assemblages.

Koch AL (2001) Oligotrophs versus copiotrophs. Bioessays 23 :657-661

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Bacteria can grow rapidly, yet there are some that grow slowly under apparent optimal conditions. These organisms are usually present in environments with low levels of nutrients, and are not found in conditions of more plentiful nutrients. They are known as "oligotrophs" in contrast to "copiotrophs", which are common in environments with greater nutritional opportunities. This essay asks why do the oligotrophs not occupy richer environments, and why are copiotrophs not more prevalent in chronic starvation environments ? BioEssays 23:657-661, 2001. (C) 2001 John Wiley & Sons, Inc.

Lage OM, Sansonetty F, O’Connor JE, Parente AM (2001) Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae. Cytometry 44 :226-235


BACKGROUND : Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells. METHODS : Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy. RESULTS : Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant. CONCLUSIONS : Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH.

Laguna R, Romo J, Read BA, Wahlund TM (2001) Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi. Appl Environ Microbiol 67 :3824-3831


Emiliania huxleyi is a unicellular marine alga that is considered to be the world’s major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.

Larsen A, Castberg T, Sandaa RA, Brussaard CPD, Egge J, Heldal M, Paulino A, Thyrhaug R, van Hannen EJ, Bratbak G (2001) Population dynamics and diversity of phytoplankton, bacteria and viruses in a seawater enclosure. Marine Ecology-Progress Series 221 :47-57

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We now know that the abundance of free viruses in most marine environments is high. There is still, however, a lack of understanding of their occurrence and distribution and of in situ relationships between viral and host communities in natural environments. This may be partly due to methodological limitations. Our main aim was therefore to perform a case study in which a variety of methods were applied in order to give an improved, high-resolution description of the microbial communities in a natural environment, In order to do this we combined light microscopy (LM), transmission electron microscopy (TEM), flow cytometry (FCM), PCR denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) and studied the diversity and succession of algae, bacteria and viruses in a nutrient enriched seawater enclosure. In the enclosure we experienced a situation where the development of the dominating algal population, which consisted of several flagellate species, was followed by proliferation of several different size-classes of viruses. The total bacterial number decreased markedly during the flagellate bloom but the community composition was maintained and the diversity remained high. Our results indicate a close linkage between various algal, bacterial and viral populations and show that virioplankton do not necessarily terminate algal and bacterial blooms but that they keep the host populations at non-blooming levels.

Lebaron P, Servais P, Agogue H, Courties C, Joux F (2001) Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems ? Appl Environ Microbiol 67 :1775-1782


The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Lebaron P, Servais P, Troussellier M, Courties C, Muyzer G, Bernard L, Schafer H, Pukall R, Stackebrandt E, Guindulain T, Vives-Rego J (2001) Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms : changes in abundances, activity and composition. Fems Microbiology Ecology 34 :255-266

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Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma -Proteobacteria, alpha -Proteubacteria were preserved, It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation. (C) 2001 Federation of European Microbiological Societies, Published by Elsevier Science B.V. All rights reserved.

Legendre L, Courties C, Troussellier M (2001) Flow cytometry in oceanography 1989-1999 : Environmental challenges and research trends. Cytometry 44 :164-172

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Background : The present review is based on the identification of four major environmental crises that have been approached from a biological oceanographic viewpoint. These crises are the release of contaminants in nearshore marine waters, the collapse of marine resources that were renewable until recently, the loss of biodiversity, and global climate change
Methods : The review examines the contribution of cytometry-based biological oceanography to the resolution of the four environmental crises. Using a database of 302 papers, flow cytometric (FCM) studies in biological oceanography over the 1989-1999 decade are examined. Future biological oceanographic applications of FCM are discussed.
Results : Most of the published FCM oceanographic studies focus on phytoplankton and bacterioplankton. Analysis of our 1983-1999 database shows the predominance of studies dedicated to phytoplankton (77%), followed by heterotrophic bacteria (21%). The latter progressively increased over the last decade, together with the improved understanding of the biogeochemical and trophic roles of marine bacteria. Most studies on these two microorganisms were conducted in vitro until 1996, after which the trend reversed in favor of in situ research. The most investigated areas were these with major international sampling efforts, related to the changing climate. Concerning environmental topics, 62% of papers on phytoplankton and bacterioplankton focused on the structure of microbial communities and fluxes (e.g., production grazing) ; this provides the basis for biological oceanographic studies on resources and climate change.
Conclusions : Future progress in the biological oceanographic use of FCM will likely fall into two categories, i.e., applications where FCM will be combined with the development of other methods and those where FCM will be the main analytical tool. It is expected that FCM and other cytometric approaches will improve the ability of biological oceanograhy to address the major environmental challenges that are confronting human societies. (C) 2001 Wiley Liss, Inc.

Legendre L, Courties C, Troussellier M (2001) Flow cytometry in oceanography 1989—1999 : environmental challenges and research trends. Cytometry 44 :164-172


BACKGROUND : The present review is based on the identification of four major environmental crises that have been approached from a biological oceanographic viewpoint. These crises are the release of contaminants in near shore marine waters, the collapse of marine resources that were renewable until recently, the loss of biodiversity, and global climate change METHODS : The review examines the contribution of cytometry-based biological oceanography to the resolution of the four environmental crises. Using a database of 302 papers, flow cytometric (FCM) studies in biological oceanography over the 1989—1999 decade are examined. Future biological oceanographic applications of FCM are discussed. RESULTS : Most of the published FCM oceanographic studies focus on phytoplankton and bacterioplankton. Analysis of our 1989-1999 database shows the predominance of studies dedicated to phytoplankton (77%), followed by heterotrophic bacteria (21%). The latter progressively increased over the last decade, together with the improved understanding of the biogeochemical and trophic roles of marine bacteria. Most studies on these two microorganisms were conducted in vitro until 1996, after which the trend reversed in favor of in situ research. The most investigated areas were those with major international sampling efforts, related to the changing climate. Concerning environmental topics, 62% of papers on phytoplankton and bacterioplankton focused on the structure of microbial communities and fluxes (e.g., production, grazing) ; this provides the basis for biological oceanographic studies on resources and climate change. CONCLUSIONS : Future progress in the biological oceanographic use of FCM will likely fall into two categories, i.e., applications where FCM will be combined with the development of other methods and those where FCM will be the main analytical tool. It is expected that FCM and other cytometric approaches will improve the ability of biological oceanography to address the major environmental challenges that are confronting human societies.

Leighfield TA, Van Dolah FM (2001) Cell cycle regulation in a dinoflagellate, Amphidinium operculatum : identification of the diel entraining cue and a possible role for cyclic AMP. J Exp Mar Bio Ecol 262 :177-197


This research describes the diel phasing of the cell cycle in the dinoflagellate, Amphidinium operculatum Claparede and Lachmann, and investigates the mechanisms that serve to link the cell cycle to the diel cycle. Unlike many dinoflagellates, A. operculatum has a naturally high division rate of approximately 1 division day(-1), which yields a nearly synchronous population, making it useful for population studies. When grown on a 16:8 h light/dark cycle, S-phase begins 10 h and mitosis 14-16 h after the onset of light, as determined by flow cytometry. Alterations in the timing of the dark/light and light/dark transitions showed that the cell cycle is entrained by the dark/light transition, with the light/dark cue being uninvolved. Cells in logarithmic phase growth also undergo diel changes in cell size (9-14 &mgr;m), reaching a maximum size late in the light phase, concurrent with mitosis. Stationary phase cells or cells blocked in G1 of the cell cycle with a cell cycle inhibitor, olomoucine, showed no size changes or reduced size changes over the diel cycle, suggesting a coupling of cell size to the cell division cycle. In Euglena, cAMP-dependent signaling appears to mediate diel phasing of the cell cycle. Therefore, the role of cAMP in cell cycle control in A. operculatum was investigated. Measurement of intracellular cAMP by radioimmunoassay (RIA) revealed that cAMP concentrations varied on a diel basis, but increases observed appeared to correlate with cell size increases, and did not correlate with light cues at the dark/light or light/dark transition. However, when cells were treated with the cAMP phosphodiesterase inhibitor, IBMX, cell cycle progression was inhibited at both the G1/S and the G2/M phase transitions. This supports a role for cAMP-dependent signaling in the dinoflagellate cell cycle and is in agreement with the documented role of cAMP in the cell cycle control of higher eukaryotes.

Lemarchand K, Parthuisot N, Catala P, Lebaron P (2001) Comparative assessment of epifluorescence microscopy, flow cytometry and solid-phase cytometry used in the enumeration of specific bacteria in water. Aquatic Microbial Ecology 25 :301-309

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Rapid microbiology and the detection of rare events are important challenges in various fields of aquatic microbiology. Epifluorescence microscopy, flow cytometry and solid-phase cytometry are techniques used for direct methods in microbiology but the range of application of these different instruments is not clearly defined. In this study, we examined the lower limit of bacterial concentration to which each technique can be reliably used. Techniques were compared for the enumeration of (1) fluorescent beads, (2) labeled bacteria at different ratios of labeled/non-labeled cells, (3) Escherichia coli O157:H7 cells inoculated at different densities in tap water, and (4) E. coli O157:H7 cells in artificially contaminated natural seawater. The different methods gave results that correlated well despite the presence of a significant background of unlabeled cells. However, solid-phase cytometry was the only technique that allowed the accurate enumeration of rare events (down to 1 cell) providing the same sensitivity as traditional culture methods. The detection sensitivity was not affected by the presence of up to 10(7) unlabeled cells on the filter. In contrast, flow cytometry was a very rapid and accurate method but it could not be applied to the detection of rare events. E. coli O157:H7 cells could be detected rapidly and accurately in environmental water samples in the presence of non-specific bacteria. Solid-phase cytometry combined with taxonomic probes allowed rapid and accurate detection of a large variety of species of ecological interest in a wide variety of aquatic environments.

Li WKW, Dickie PM (2001) Monitoring phytoplankton, bacterioplankton, and virioplankton in a coastal inlet (Bedford Basin) by flow cytometry. Cytometry 44 :236-246

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Background : To establish the prevailing state of the ecosystem for the assessment of long-term change, the abundance of microbial plankton in Bedford Basin (Nova Scotia, Canada) is monitored weekly by flow cytometry.
Methods : Phytoplankton are detected by their chlorophyll autofluorescence. Those that contain phycoerythrin are designated as Synechococcus cyanobacteria or cryptophyte algae according to the intensity of light scatter. Bacteria and viruses are stained with DNA-binding fluorochromes and detected by green fluorescence. Distinction is made between bacterial and vital subpopulations exhibiting high and low fluorescence.
Results : Time series data are presented for weekly observations from 1991 to 2000. Weekly averages are computed for the complete annual cycle of temperature, salinity, river discharge, nitrate, phosphate, silicate, chlorophyll, total phytoplankton including Synechococcus and cryptophytes, total bacteria including high and low-fluorescence subpopulations, and total viruses including high and low-fluorescence subpopulations.
Conclusions : The microbial biomass in the surface water of Bedford Basin is dominated by phytoplankton The spring bloom of phytoplankton represents a maximum in algal biovolume, but not in cell number. Phytoplankton, bacteria, and viruses all attain their annual numerical maxima between the summer solstice and the autumn equinox. A vigorous microbial loop and viral shunt is envisioned to occur in the summer. (C) 2001Wiley-Liss, Inc.

Li WKW, Harrison WG (2001) Chlorophyll, bacteria and picophytoplankton in ecological provinces of the North Atlantic. Deep-Sea Research Part Ii-Topical Studies in Oceanography 48 :2271-2293

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A comparative ecology of chlorophyll, bacteria and picophytoplankton is presented for seven ecological provinces in the North Atlantic. Depth-integrated standing stocks of these biota were measured from boreal polar to subtropical gyral regions. Averaging over all sampling times and locations within each province, it appeared that the integrated biomass of bacteria did not exceed that of phytoplankton in any province. Although this biomass ratio often exceeded unity in surface waters of the subtropical gyral provinces, the ratio for the upper water column as a whole was lowered by the subsurface chlorophyll layer. Bacteria and picophytoplankton, as the potential food resource of micrograzers, appeared to complement each other such that their total biomass did not vary much more than 2-fold amongst the seven provinces. Characteristic parameters of the biotic depth profiles, namely surface concentrations, integrated stocks and depth of maximum, were used to cluster the provinces. The original classification of provinces based on surface chlorophyll fields and characteristic regional physics was reinforced by the inclusion of bacteria and picophytoplankton. Crown Copyright (C) 2001 Published by Elsevier Science Ltd. All rights reserved.

Marie D, Partensky F, Vaulot D, Brussaard C (2001) Enumeration of phytoplankton, bacteria, and viruses in marine samples. Curr Protoc Cytom Chapter 11 :Unit 11 11


For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.

Marie D, Simon N, Guillou L, Partensky F, Vaulot D (2001) DNA/RNA analysis of phytoplankton by flow cytometry. Curr Protoc Cytom Chapter 11 :Unit 11.12

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The past 10 years or so have seen the combination of molecular and biochemical techniques within the confines of cytometry. The use of flow cytometry in microbiology is finally coming of age. This unit carefully defines the criteria for evaluation of DNA and RNA in phytoplankton. Of course not everyone works with phytoplankton, but the methods outlined are very appropriately representative for other organisms. In addition, the unit discusses the methods for evaluating cell cycle and discriminating specific taxa using fluorescent oligonucleotide probes targeted to 18S rRNA.

Massana R, Pedros-Alio C, Casamayor EO, Gasol JM (2001) Changes in marine bacterioplankton phylogenetic composition during incubations designed to measure biogeochemically significant parameters. Limnology and Oceanography 46 :1181-1188

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Bottle incubations, during which the activity and growth of prokaryotes is monitored during several days, are frequently carried out to study functional aspects of marine prokaryotic assemblages. These experiments will relate directly to in situ activities if all populations grow harmonically during the incubation. We tested whether this was the case by analyzing the composition of bacterial assemblages at the beginning and at the end of the incubation by denaturing gradient gel electrophoresis. Five experiments were done in different Antarctic regions. Bacterial assemblages north and south of the Polar Front were very different. In all cases, the final assemblages were very different from the initial ones, and these changes were often accompanied by a significant decrease of diversity indices. Our experiments included treatments with different temperature and organic matter amendments. Whereas the increase in temperature tested had a minor effect on prokaryotic growth rate and specific composition, the addition of organic matter strongly stimulated growth rate and selected a particular bacterial assemblage in some experiments but not in others. A significant component of bacterial assemblages From waters south of the Polar Front appeared to be Polari-bacter franzmannii, a gas vacuolated bacterium of the Cytophaga-Flavobacterium-Bacteroides group that was originally isolated from Antarctic sea ice. This phylotype was enriched and dominated in almost all final assemblages. Our results indicate that long-term bottle incubations mostly measure the activity of a few opportunistic bacteria and not that of the original assemblage. This should be taken into account if data obtained in these experiments are used for balancing whole ecosystem carbon budgets and to derive biogeochemical conclusions.

Micic M, Bihari N, Labura Z, Muller WE, Batel R (2001) Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri-n-butyltin chloride. Aquat Toxicol 55 :61-73


Induction of apoptosis by tri-n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1-5 microg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G(0)/G(1) cell cycle region (the sub-G(1) region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels’ gills was indicated by the selective loss of G(2)/M cells concomitant with the appearance of cells with decreased DNA content in S and G(0)/G(1) cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.

Moreira-Turcq PF, Cauwet G, Martin JM (2001) Contribution of flow cytometry to estimate picoplankton biomass in estuarine systems. Hydrobiologia 462 :157-168

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Picoplankton (plankton greater than or equal to3 mum) biomass was determined by flow cytometry in three European estuarine systems (Krka Estuary in Croatia, Rhone Delta in France, and Lena Delta and Laptev Sea in Russia). The size of natural phytoplankton groups was obtained by a calibration curve, with different picoplankton’s strains (from 1.6 to 3.4 mum), measured by a Coulter counter (size) and a flow cytometer(light-scattering). Two natural groups of picoplankton were identified by flow cytometry in the three systems : Synechococcus sp and picoeukaryotes. Picoplankton cells abundance ranged between : 2800 and 42 000, 5000 and 37 000, 1000 and 50 000 cells ml(-1) in the Krka estuary, in the Rhone delta and in the Lena-Laptev system, respectively. In the Krka estuary, picoplankton biomass ranges between 11 and 68 mugC l(-1). It can make up as much as 88% of the total photosynthetic plankton population and 50% of total organic particulate carbon. Picoplankton biomass was greater in the summer than in the autumn. At the halocline layer this biomass can attempt ca. 390 mugC l(-1) during the summer cruise. In the Rhone delta, a lower picoplankton biomass (6-39 mugC l(-1)) was observed at the end of the winter. These biomass represented between 0.4 and 22% of the particulate organic carbon, which could reach 71% of the total photosynthetic plankton biomass at the marine station. In the Lena-Laptev system, picoplankton biomass varied between 6 and 56 muC l(-1) in surface waters. Picoplankton biomass decreased with depth, but picoeukaryotes were still observed in deep samples (20, 30 m) in the Laptev Sea, showing a considerable autotrophic activity in spite of low temperatures (0-1degreesC). Although the widely dispersed estuary geographic distribution and their different estuarine characteristics, the data point out that these small organisms can also play an important role in the transfer of organic carbon from rivers to oceans and that flow cytometry can be able to detect these small cells in turbid systems.

Moreno-Garrido I, Hampel M, Lubian LM, Blasco J (2001) Marine microalgae toxicity test for linear alkylbenzene sulfonate (LAS) and alkylphenol ethoxylate (APEO). Fresenius J Anal Chem 371 :474-478


Different microalgal species have been used in growth-inhibition tests to determine the toxic concentrations of anionic and non-ionic surfactants to phytoplankton. The species used were selected from different taxonomic groups, all of considerable ecological relevance to marine environments. The toxicity of the C13 LAS homologue to the microalgal species selected was usually one order of magnitude greater than that of the C11 homologue. The toxicity of a commercial LAS mixture to different microalgal species was also checked. For this material and C. gracilis, cellular counting by means of a Neubauer chamber and by use of a flow cytometer were compared ; differences between the two methods were insignificant. The toxicity of decaethoxylated nonylphenol non-ionic surfactant to C. gracilis was also checked ; the EC50 value for this compound was 1.0 mg L(-1).

Morris CW, Autret A, Boddy L (2001) Support vector machines for identifying organisms - a comparison with strongly partitioned radial basis function networks. Ecological Modelling 146 :57-67

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Biological identification poses many problems which are not commonly met in other areas where artificial neural networks (ANNs) are applied. One such problem is that the number of possible classes is often unbounded and it is necessary to be able to deal with novel categories (taxa) either by rejecting them or retraining to allow for them. Where it is necessary to incorporate them, retraining a neural network may be time consuming and or impossible. One way of negating this problem is to use single-category networks that can be combined and configured as required.
This paper compares support vector machines (SVMs) with strongly partitioned traditional radial basis function (RBF) networks for the discrimination of single species of phytoplankton against a background of N other species. SVMs resulted in greater identification success than the unpartitioned, large single RBF networks (81 and 77%, respectively), though partitioned RBF ANNs performed relatively poorly (50% success) at discrimination of 61 marine phytoplankton species from flow cytometry data. Greatest success was achieved by combining the outputs of the individual networks by means of a ’winner takes all’ strategy ; with RBF ANNs identification success dramatically increased (from 16 to 50%), though there was only a modest increase with SVMs (77-81%). When SVMs trained on one data set were tested with data on cells grown under different light conditions, an overall successful identification was low (13%), but when SVMs were trained on a combined data set identification was high (> 70%). Clearly, it is essential to cover the whole spectrum of biological variation in the training data set. (C) 2001 Elsevier Science B.V. All rights reserved.

Mulero V, Pelegrin P, Sepulcre MP, Munoz J, Meseguer J (2001) A fish cell surface receptor defined by a mAb mediates leukocyte aggregation and deactivation. Developmental and Comparative Immunology 25 :619-627

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Cell adhesion molecules play a key role in the inflammatory response. Selectins, integrins and immunoglobulin gene superfamily adhesion receptors mediate the different steps of leukocyte migration from the blood-stream towards inflammatory foci. In addition to their adhesive function, these receptors modulate major cellular processes such as cell activation, growth, differentiation and death. To characterise the fish molecules involved in cell adhesion, a panel of mAbs was raised by immunising mice with macrophages from the marine fish gilthead seabream (Sparus aurata L.). One of these mAbs, which we named anti-Aggregatin, was found to induce a rapid heterotypic aggregation of seabream leukocytes. Anti-Aggregatin defined a 140-kDa cell surface receptor which was highly expressed by macrophages and was up-regulated after co-stimulation with LPS and MAF. Functionally, the cell adhesion which occurred upon exposure to anti-Aggregatin required Ca2+, an intact cytoskeleton and an active cell metabolism. More importantly, Aggregatin engagement resulted in strong inhibition of the phagocyte respiratory burst, although the cells showed neither loss of viability nor DNA fragmentation. The results are discussed in relation to the potential role of cell adhesion molecules in fish immune responses. (C) 2001 Published by Elsevier Science Ltd.

Muller S, Kiesel B, Berthe-Corti L (2001) Muricauda ruestringensis has an asymmetric cell cycle. Acta Biotechnologica 21 :343-357

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Muricauda ruestringensis B1 is a GRAM-negative, marine bacterium and a member of the Flavobacteriaceae family. It is characterized by long appendages, which appear at different stages of growth. At the outer end of these appendages there is a bulbous structure. Investigating the cell morphology of strain B1 during batch growth revealed a high diversity of cell types and sizes. Apart from small rod-shaped cells and rods with appendages, there were large rods and spherical cells of different sizes as well as spherical cells which had fimbriae. To be able to study the cell cycle events, it was essential to monitor the population dynamics of the involved individuals. For this purpose, fluorochromising techniques, multi-parametric flow cytometry, image analysis and fluorescence microscopy were used. It was demonstrated that all cell types displayed a broad variation in DNA content, the precise number of chromosomes varied depending on the growth phase. The assortment was testified to hold 16S rDNA sequence identity. The cultures consisted of subpopulations whose density within a Percoll gradient varied considerably, ranging from 1.028 to 1.070. Consolidating the results of the morphological data, the chromosome content and the density of the subpopulations at different growth stages enabled us to construct an asymmetric cell cycle for the growth of strain B1 under the specific culture conditions of our experiments.

Palenik B (2001) Chromatic adaptation in marine Synechococcus strains. Applied and Environmental Microbiology 67 :991-994

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Characterization of two genetically distinct groups of marine Synechococcus sp. strains shows that one, but not the other, increases its phycourobilin/phycoerythrobilin chromophore ratio when growing in blue light. This ability of at least some marine Synechococcus strains to chromatically adapt may help explain their greater abundance in particular ocean environments than cyanobacteria of the genus Prochlorococcus.

Pendleton A, Koffer A (2001) Effects of latrunculin reveal requirements for the actin cytoskeleton during secretion from mast cells. Cell Motil Cytoskeleton 48 :37-51


To investigate the role of the actin cytoskeleton in exocytosis, we have tested the effects of latrunculin B, a microfilament-disrupting drug, on secretion from intact and permeabilised rat peritoneal mast cells. The toxin strongly inhibited secretion from intact cells (attached or in suspension) responding to a polybasic agonist, compound 48/80. However, this effect was revealed only after a profound depletion of actin filaments. This was achieved by a long (1 h) exposure of cells to the drug before activation, together with its presence during activation. Maximal inhibition of secretion by such treatment was 85% at 40 microgram/ml latrunculin B. These results indicate that minimal actin structures are essential for the exocytotic response. In contrast, stimulus-induced cell spreading was prevented by latrunculin (5 microgram/ml) applied either before or after activation. The effects of the toxin on intact cells were fully reversible. The responses of permeabilised cells were affected differentially : secretion induced by calcium was more sensitive to latrunculin than that induced by GTP-gamma-S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton than the response induced by GTP-gamma-S. Again, maximal inhibitory effects (approximately 65 and 25% at 40 microgram/ml) were observed only when cells were exposed to the toxin both before and after permeabilisation. Since the permeabilised cells system focuses on the final steps of exocytosis, the incomplete inhibition suggests that actin plays a modulatory rather than a central role at this stage.

Perez-Prieto S, Garcia-Rosado E, Rodriguez S, Castro D, Borrego JJ (2001) Antigenic properties and experimental transmission to several fish species of a marine birnavirus isolated from sole (Solea senegalensis). Vet Microbiol 82 :11-25


A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the solevirus to other fish species has been determined by inoculation to freshwater and marine fish species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the marine fish species, although solevirus caused an asymptomatic infection in all species tested, as demonstrated by the detection of viral RNA and of viral antigens in fish leucocytes, respectively, using polymerase chain reaction (PCR) and flow cytometry (FC).

Pernthaler A, Pernthaler J, Eilers H, Amann R (2001) Growth patterns of two marine isolates : adaptations to substrate patchiness ? Appl Environ Microbiol 67 :4077-4083


During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus Pseudoalteromonas rapidly multiplied in substrate-amended North Sea water, whereas the densities of Oceanospirillum changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634-4640, 2000). We therefore studied the growth patterns of two isolates affiliating with Pseudoalteromonas and Oceanospirillum in batch culture. Upon substrate resupply, Oceanospirillum lagged threefold longer than Pseudoalteromonas but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved Oceanospirillum populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of Oceanospirillum were reduced three times as much as those Pseudoalteromonas, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the Pseudoalteromonas population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.

Rajbhandari I, Takamatsu S, Nagle DG (2001) A new dehydrogeranylgeraniol antioxidant from Saururus cernuus that inhibits intracellular reactive oxygen species (ROS)-catalyzed oxidation within HL-60 cells. J Nat Prod 64 :693-695


A new triene, 12,13-dehydrogeranylgeraniol (1), was isolated from the aquatic plant Saururus cernuus and its structure determined spectroscopically. Compound 1 inhibits PMA-induced peroxide-catalyzed oxidation of 2’,7’-dichlorodihydrofluorescein dye (DCFH) by reactive oxygen species (ROS) within human promyelocyctic HL-60 cells.

Resina-Pelfort O, Comas-Riu J, Vives-Rego J (2001) Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton. Syst Appl Microbiol 24 :31-36


The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.

Serra T, Colomer J, Cristina XP, Vila X, Arellano JB, Casamitjana X (2001) Evaluation of laser in situ scattering instrument for measuring concentration of phytoplankton, purple sulfur bacteria, and suspended inorganic sediments in lakes. Journal of Environmental Engineering-Asce 127 :1023-1030

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A laser in situ scattering and transmissometry (Lisst-100) probe has been used for estimating the particle-size distribution of phytopankton, purple photosynthetic sulphur bacteria (Chromatiaceae), and suspended inorganic sediments in different lakes. Results from Lisst-100 have been compared to laboratory measurements, such as those obtained by using a Galai laser size analyzer (GL), an optical microscope (OM), and a flow cytometer (FC). Although all of these instruments were shown to provide reliable values of the particle number concentration for the given populations, the Lisst-100 was the fastest and most reliable instrument because it did not require manipulation of the samples-which is not the case of GL, OM and FC instruments -and avoided the tedious procedure of microscopic counts. The total particle volume concentration results obtained with Lisst-100 differed from those obtained with GL for populations with large and porous aggregates, such as phytoplankton cells. The difference was attributed to the breakage of fragile algal aggregates resulting from the measuring procedure used by GL. Although for suspended sediment particles both instruments gave the same results for the total particle volume concentration, the particle-size distribution obtained with GL was found always shifted to smaller diameters than with Lisst-100, probably because inorganic sediment particles present compact aggregates. When these aggregates break, they split into a high number of small particles that contribute the same to the total volume concentration as the previous aggregates. Finally, results of the total particle volume concentration with Lisst-100 were in accordance with those obtained with GL for the Chromatiaceae population, because cells remained in a dispersed phase. A good correlation was found between the total particle volume concentration of Chromatiaceae measured with Lisst-100 and the concentration of bacteriochlorophyll a (BChl a), which is the parameter habitually used to estimate the concentration of Chromatiaceae. Therefore, Lisst-100 was found to be a reliable instrument to estimate the Chromatiaceae concentration in aquatic ecosystems.

Servais P, Agogue H, Courties C, Joux F, Lebaron P (2001) Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments ? FEMS Microbiol Ecol 35 :171-179


The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

Sherry ND, Wood AM (2001) Phycoerythrin-containing picocyanobacteria in the Arabian Sea in February 1995 : diel patterns, spatial variability, and growth rates. Deep-Sea Research Part Ii-Topical Studies in Oceanography 48 :1263-1283

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The abundance of phycoerythrin-containing picocyanobacteria in the surface mixed layer was measured both along-shore and offshore between 8 and 23 February 1995 in the Northwestern Arabian Sea. Water samples from 3m depth were taken at 2-h intervals and picocyanobacterial abundance and frequency of dividing cells were determined by epifluorescence microscopy, Cell counts showed an average diel change from a mid-day minimum of similar to 50 x 10(3) cells ml(-1) to an evening maximum of similar to 180 x 10(3) cells ml(-1). The diel change was greater than the differences observed between physically and spatially discrete water masses. By counting the frequency of dividing cells (FDC) and using a novel approach to estimating the length of time required to complete cell division, growth and loss rates were both estimated to be similar to 2.9 d(-1) with daily turnover being 140% of the mean standing stock. If differences in the intrinsic population growth rate (mu) and the net rate of change in cell number (r) are assumed to be due to grazing, then grazing occurred throughout the day at a relatively constant rate (reflecting phytoplankton loss rates of similar to 0.12h(-1)). Cell division rates peaked in the late afternoon and early evening. FDC decreased throughout the night, suggesting that dark-inhibition of cell division is weak or nonexistent in the picocyanobacteria we studied. While all cell types included in this study would be identified as Synechococcus by flow cytometry because they were small unicells with bright phycoerythrin fluorescence, morphological variability suggests that the community was actually taxonomically diverse and included cells other than Synechococcus, including Synechocystis. Despite this diversity, the strong diel patterns we observed persisted throughout the study region, suggesting that great care should be taken when interpreting picocyanobacterial survey data and experimental results that do not account for the effects of time-of-day. (C) 2001 Elsevier Science Ltd. All rights reserved.

Simo R (2001) Production of atmospheric sulfur by oceanic plankton : biogeochemical, ecological and evolutionary links. Trends in Ecology & Evolution 16 :287-294

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Biological production of the volatile compound dimethylsulfide in the ocean is the main natural source of tropospheric sulfur on a global scale, with important consequences for the radiative balance of the Earth. In the late 1980s, a Gaian feedback link between marine phytoplankton and climate through the release of atmospheric sulfur was hypothesized. However, the idea of microalgae producing a substance that could regulate climate has been criticized on the basis of its evolutionary feasibility. Recent advances have shown that volatile sulfur is a result of ecological interactions and transformation processes through planktonic food webs. It is, therefore, not only phytoplankton biomass, taxonomy or activity, but also food-web structure and dynamics that drive the oceanic production of atmospheric sulfur. Accordingly, the viewpoint on the ecological and evolutionary basis of this amazing marine biota-atmosphere link is changing.

Sondergaard M, Danielsen M (2001) Active bacteria (CTC+) in temperate lakes : temporal and cross-system variations. Journal of Plankton Research 23 :1195-1206

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The temporal variation in the abundance and proportion of highly respiration-active bacteria in the eutrophic lakes Esrum and Frederiksborg Slotsso was determined with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In addition, a comparative late summer study was undertaken across a gradient of nutrient enrichment in Danish lakes. The purpose was to investigate the importance of substrate (chlorophyll) and temperature for the control of CTC-active cells (CTC+). The abundance of CTC+ cells was much lower and more variable than the total number of cells counted after 4’,6-diamidino-2-phenylindole (DAPI) staining. The proportion of CTC+ cells in Lake Esrum and Frederiksborg Slotsso was normally <5%, and between 2.5 and 20% in 14 other lakes. The abundance as well as the proportion of CTC+ cells increased with chlorophyll in Lake Esrum and Frederiksborg Slotsso, and chlorophyll explained 43% of the variability in CTC+ abundance. In the comparative study, the abundance of CTC+ cells increased along the chlorophyll gradient, which explained 49% of the variability. The results showed that the abundance and, to a lesser degree, the proportion of CTC+ bacteria were controlled by substrate supply. One consequence of the low abundance of active bacteria is that in situ growth rates scaled to CTC+ cells are 3- to 7-fold higher than those scaled to DAPI counts. It is suggested that studies on factors controlling bacterioplankton activity at the single-cell level should be investigated scaled to active cells.

Theilacker GH, Shen W (2001) Evaluating growth of larval walleye pollock, Theragra chalcogramma, using cell cycle analysis. Marine Biology 138 :897-907

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Cell cycle analysis of muscle cell division rates offers a new and efficient technique to analyze growth of larval fish. Using this approach, growth of larval walleye pollock was estimated by determining cell proliferation rates, reasoning that growth during early life stages is probably attributed to increases in cell number rather than to increases in cell size. Characteristic patterns of brain and muscle cell division rates were produced in larval walleye pollock by manipulating their diet in the laboratory. The fraction of dividing muscle cells and, to a lesser extent, the fraction of dividing brain cells were direct indicators of fast and slow growth. A model was produced to estimate average growth rate from the fraction of dividing muscle cells. We developed a simple method for preparing and storing the muscle tissue that ensures nucleic acid stability for subsequent analyses and permits sampling in the field. We envision that the cell cycle methodology will have on-site applications, presenting an opportunity to attain real-time estimates of larval fish growth at sea. Determining the proportion of first-feeding larvae with a high fraction of dividing muscle cells may yield a means for predicting the proportion of fast-growing fish, i.e., the potential survivors.

Uysal Z (2001) Chroococcoid cyanobacteria Synechococcus spp. in the Black Sea : pigments, size, distribution, growth and diurnal variability. Journal of Plankton Research 23 :175-189

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Phycoerythrin-containing unicellular cyanobacteria Synechococcus spp. were studied for the first time during April-May 1994 and September-October, 1996, in the western and southern Black Sea for pigments, size and abundance distribution via spectrometry, epifluorescence microscopy and flow cytometry. Abundance distribution in the surface mixed layer in April-May, 1994, revealed that cells were more concentrated in offshore waters than in coastal regions under the direct influence of the river Danube. However, in the south, higher surface cell concentrations were characteristic of the nearshore areas during September-October, 1996. A highly significant correlation was observed between cell abundance and ambient physico-chemical parameters with depth. Visual inspection of the individual cells under the epifluorescence microscope revealed that cells at the subsurface, chlorophyll a maximum layer (SCML, based on in situ fluorometer readings) fluoresce more brightly and for longer than those at the surface and at lower depths. Spectral properties of a total of 64 Synechococcus spp. clonal isolates from different depths within the euphotic layer (about the top 60 m) in the southern Black Sea coast showed that all have type 2 phycoerythrobilin in common, lacking phycourobilin. In vivo fluorescence emission maxima for phycoerythrobilin were about the same (similar to 578 nm) for all isolates. All isolates had in vivo absorption maxima at between 435 and 442 nm, and at about 681 nm due to chlorophyll a. It was shown from the flow cytometer mean forward light scatter data for size distribution that cells at the surface mixed layer (0-10 m) were larger than cells at lower depths (20-60 m). Based on in vivo fluorescence measurements, significant differences in the acclimated growth rates of clones from different depths were observed. Time versus cell count plots showed that cells of the cyanobacteria Synechococcus spp. are under grazing pressure, from midnight until noon, and slowly begin to rebuild their population in the afternoon by dividing throughout the evening.

Vaque D, Casamayor EO, Gasol JM (2001) Dynamics of whole community bacterial production and grazing losses in seawater incubations as related to the changes in the proportions of bacteria with different DNA content. Aquatic Microbial Ecology 25 :163-177

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Natural bacterial assemblages are not physiologically and phenotypically homogeneous. Development of new methodologies, such as flow cytometry, has allowed bacterial types with different degrees of activity (which have been called HighDNA and LowDNA bacteria) to be distinguished on the basis of their DNA content. Because previous data have suggested that HighDNA bacteria are really the active fraction of the community, we hypothesize that the dynamics of bacterial production (BP) and grazing losses should be linked to the changes of this fraction rather than to changes in the whole community. To test our hypothesis we took samples during a cruise in the NW Mediterranean Sea in March 1999 from 6 selected stations placed along 2 transects, 1 perpendicular to the city of Barcelona and the other to Palamos, In each transect we visited ’coastal’ (on the continental platform), ’slope’ and ’open sea’ (> 2000 m) stations. Samples were collected at surface and at the deep chlorophyll maximum. Bacterial abundance (total, HighDNA and LowDNA) and BP were determined in in situ samples. Also, 12 experiments were performed to survey the dynamics of HighDNA bacteria (percentage and biomass), BP and grazing rates inside experimental bottles every 8 to 12 h for a total period of 44 h. Bacterial abundance was counted and cell volume was estimated by flow citometry, BP was determined by H-3-leucine incorporation, and grazing rate was obtained by following the evolution inside the experimental bottles of added 5-([4,6 dichlorotriazin-2yl) amino] -fluorescein-stained Pseudomonas diminuta as fluorescent-labeled bacteria. In situ, BP was higher in coastal and slope stations than in open sea stations. The percentage of HighDNA bacteria ranged between 25 and 87 % and BP between 0.09 and 5.9 pg C l(-1) d(-1), lower in the open sea and higher in the slope station of Palamos. Grazing loss rates followed a similar pattern, from 0.2 x 10(5) to 2.2 x 10(5) cells ingested ml(-1) d(-1), again lower in the open sea and higher at the coastal station of Palamos. In most of the experiments, BP increased with time following the increase of HighDNA bacteria, while LowDNA bacteria remained practically constant during the whole period. Exponential bacterial growth appeared at 20 to 32 h. Grazing rates were maximal right after the exponential bacterial growth (at 32 to 44 h), suggesting that the increase of the HighDNA bacterial fraction in the previous period stimulated grazers to consume it. In both in situ and experimental samples, abundance and biomass of HighDNA bacteria were strongly correlated with BP and grazing rates, but no correlation was found between these variables and LowDNA bacterial abundance. Samples with the lowest percentage of HighDNA bacteria also had low BP and grazing rates. Indeed, the dynamics of BP and grazing losses were better related to changes in HighDNA bacteria, providing further evidence that they can be considered as the ’active’ fraction of the whole bacterial community.

Veldhuis MJW, Kraay GW, Timmermans KR (2001) Cell death in phytoplankton : correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. European Journal of Phycology 36 :167-177

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Different stages of the automortality in phytoplankton have been studied applying flow cytometry. These stages are, in order of expression : (1) compromised cell membranes, (2) degradation of the photosynthetic pigments and reduction of the photosynthetic activity, (3) fragmentation of the genomic DNA. The integrity test of the cell membranes is based on the inability of the DNA-specific stain SYTOX Green to pass into cells with intact plasma membranes. The reduction in photosynthetic activity was examined by sorting C-14-labelled phytoplankton cells differing in viability. Finally, DNA fragmentation was traced by measuring changes in genomic DNA. The different phytoplankton species tested showed a great variety in response when grown under the same conditions, but there was also considerable intraspecific variation. Unstained cells, fully stained cells (equivalent to full staining of genomic DNA in fixed cells) and cells with intermediate fluorescence signal occurred together within the same culture. The photosynthetic activity in cells with a reduced viability dropped by as much as 60 % relative to that of the viable cells. In the subsequent stage, when photosynthetic pigments were fully degraded, this value dropped further to around 10 %. Cells in this stage also showed subdiploidy as a result of genome fragmentation. Field tests using samples of phytoplankton collected in the North Atlantic Ocean (40 degrees N, 23 degrees W) during spring showed staining properties similar to those found in cultures grown at suboptimal growth conditions. The percentage of non-viable cells varied considerably (ranging from 5 % to 60 %) between the various phytoplankton groups present. The lowest value was observed for Synechococcus, but some pico-eukaryotes showed percentages as high as 60%. Moreover, the viability varied with depth (light level) and over a light-dark cycle. The present findings suggest the existence of a (genetically based) uniform process of automortality in phytoplankton. Non-viable cells are a substantial component of the oceanic phytoplankton, affecting the food-web structure and species succession.

Wang A, Barber D, Pfeiffer CJ (2001) Protective effects of selenium against mercury toxicity in cultured Atlantic spotted dolphin (Stenella plagiodon) renal cells. Arch Environ Contam Toxicol 41 :403-409


Marine mammals are known for their low susceptibility to mercury toxicity, and selenium may play a role in this protection against mercury intoxication. To gain insight into mechanisms by which selenium might inhibit mercury toxicity in cetacean cells, we investigated the effects of sodium selenite on cell proliferation and cell death (including apoptosis, oncosis, and necrosis) of control and mercuric chloride-treated Atlantic spotted dolphin renal cells (Sp1K cells). Concurrent exposure to 80 microM Na2SeO3 provided full protection against the decrease in cell proliferation induced by 20 microM HgCl2. Pretreatment with Na2SeO3 increased the protective effects of selenium administered later in conjunction with mercury, but pretreatment alone did not provide protection against mercury given alone. Furthermore, Na2SeO3 administered after the exposure to HgCl2 did not protect cells. These data suggest that the coexistence of Na2SeO3 and HgCl2 was essential for the protective effects of Na2SeO3 against the toxicity of HgCl2 in Sp1K cells, and may involve selenium-mercury binding. This is supported by the results of an experiment in which earlier premixed mercury and selenium solutions were less cytotoxic than freshly mixed solutions. Furthermore, HgCl2 induced apoptosis in Sp1K cells, as revealed by nuclear specific dye (7-AAD) incorporation and cell flow cytometry, and this was prevented by the concurrent exposure to Na2SeO3. Inhibition of mercury-induced apoptosis in marine mammal cells, provided by selenium, may contribute to the in vivo protection. This study is the first report that addresses the mechanism of mercury-selenium antagonism in cultured cetacean cells at the cellular level.

Weisse T, Karstens N, Meyer VCL, Janke L, Lettner S, Teichgraber K (2001) Niche separation in common prostome freshwater ciliates : the effect of food and temperature. Aquatic Microbial Ecology 26 :167-179

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We characterized the ecological niches of several planktonic prostome ciliates with respect to their food demand and temperature. We found intergeneric differences between Balanion planctonicum and the 2 Urotricha spp., U. furcata and U. farcta. There were also significant interspecific differences within the genus Urotricha and intraspecific differences between 2 Balanion spp. and 3 U, furcata isolates from distant lakes. Relative to Urotricha spp., Balanion appeared to be the superior competitor at low to medium food concentrations and reached high growth rates at moderate temperatures. The threshold prey concentration for positive population growth of B. planctonicum was lower than that obtained for the 2 Urotricha spp., but higher than that reported earlier for the marine species, B. comatum. A third Urotricha species, U. castalia, was investigated for its temperature response only, The temperature response revealed species-specific temperature adaptation between B. planctonicum and the sympatric U. furcata, and further differences within the genus Urotricha : U, farcta grew fastest at high temperatures ; U. castalia was adapted to low temperatures ; and U, furcata peaked at moderately warm temperatures.

Wen R, Wang D, McKay C, Bunting KD, Marine JC, Vanin EF, Zambetti GP, Korsmeyer SJ, Ihle JN, Cleveland JL (2001) Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development. Mol Cell Biol 21 :678-689


Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

West NJ, Schonhuber WA, Fuller NJ, Amann RI, Rippka R, Post AF, Scanlan DJ (2001) Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides. Microbiology 147 :1731-1744


An in situ hybridization method was applied to the identification of marine cyanobacteria assignable to the genus Prochlorococcus using horseradish-peroxidase-labelled 16S rRNA-targeted oligonucleotide probes in combination with tyramide signal amplification (TSA). With this method very bright signals were obtained, in contrast to hybridizations with oligonucleotides monolabelled with fluorochromes, which failed to give positive signals. Genotype-specific oligonucleotides for high light (HL)- and low light (LL)-adapted members of this genus were identified by 16S rRNA sequence analyses and their specificities confirmed in whole-cell hybridizations with cultured strains of Prochlorococcus marinus Chisholm et al., 1992, Prochlorococcus sp. and Synechococcus sp. In situ hybridization of these genotype-specific probes to field samples from stratified water bodies collected in the North Atlantic Ocean and the Red Sea allowed a rapid assessment of the abundance and spatial distribution of HL- and LL-adapted Prochlorococcus. In both oceanic regions the LL-adapted Prochlorococcus populations were localized in deeper water whereas the HL-adapted Prochlorococcus populations were not only distinct in each region but also exhibited strikingly different depth distributions, HLI being confined to shallow water in the North Atlantic, in contrast to HLII, which was present throughout the water column in the Red Sea.

Wilkins MF, Hardy SA, Boddy L, Morris CW (2001) Comparison of five clustering algorithms to classify phytoplankton from flow cytometry data. Cytometry 44 :210-217


BACKGROUND : Artificial neural networks (ANNs) have been shown to be valuable in the analysis of analytical flow cytometric (AFC) data in aquatic ecology. Automated extraction of clusters is an important first stage in deriving ANN training data from field samples, but AFC data pose a number of challenges for many types of clustering algorithm. The fuzzy k-means algorithm recently has been extended to address nonspherical clusters with the use of scatter matrices. Four variants were proposed, each optimizing a different measure of clustering "goodness." METHODS : With AFC data obtained from marine phytoplankton species in culture, the four fuzzy k-means algorithm variants were compared with each other and with another multivariate clustering algorithm based on critical distances currently used in flow cytometry. RESULTS : One of the algorithm variants (adaptive distances, also known as the Gustafson—Kessel algorithm) was found to be robust and reliable, whereas the others showed various problems. CONCLUSIONS : The adaptive distances algorithm was superior in use to the clustering algorithms against which it was tested, but the problem of automatic determination of the number of clusters remains to be addressed.

Xue QG, Renault T, Chilmonczyk S (2001) Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph. Fish & Shellfish Immunology 11 :557-567

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The morphology and functions of haemocytes from the haemolymph of the European oyster, Ostrea edulis, were analysed by flow cytometry on the basis of cellular structures and incorporation of fluorescent markers. O. edulis circulating haemocytes appear to be composed of one to three cell populations based on cell size and granularity, with many individual variations. Analysis of haemocytes after propidium iodide staining indicated that the majority of oyster haemocytes are alive after sampling. The phagocytic activity level of haemocytes was analysed using fluorescent beads and this cell activity varied greatly depending on the oysters. The use of 3,3’dihexyloxacarbocyanine iodide (DIOC6) allowed the demonstration of several cell populations on the basis of labelled intensity. One to three cell sub-populations can be observed depending on the oysters. The haemocytes characterised by high granularity showed a strong fluorescence intensity related to high mitochondrial activity., (C) 2001 Academic Press.

Zhu HB, Geng MY, Li J, Guan HS (2001) Antiproliferative effects of D-polymannuronic sulfate on rat vascular smooth muscle cells and its related mechanisms. Acta Pharmacol Sin 22 :706-710


AIM : To investigate the inhibitory effects of D-polymannuronic sulfate (DPS) on the proliferation of rat vascular smooth muscle cells (VSMC) induced by angiotensin II (Ang II) and its related mechanisms. METHODS : The effects of DPS on Ang II-induced proliferation of VSMC were evaluated by MTT assay. The intracellular free Ca2+ concentrations, protein contents, and cell cycle were analyzed by flow cytometry. RESULTS : DPS 0.001 - 100 mg/L blocked the cell cycle at the G0/G1—>S transit and prevented the cells from entering into the G2/M phase, and its inhibitory effects on an increase in intracellular free Ca2+ concentrations and the protein synthesis of VSMC were also observed. Also, the suppressing actions of DPS on intracellular Ca2+ were completely blocked by L-NAME, a nitric oxide synthase inhibitor, indicating that the counteracting effects on a rise in intracellular free Ca2+ contents by DPS might be mediated by participation of NO. CONCLUSION : DPS exerted an inhibitory effect on Ang II-induced proliferation of VSMC and its related mechanisms were considered to be related to its inhibition on the increment of intracellular Ca2+ concentrations, which subsequently suppressed the synthesis of DNA and protein of VSMC.

Ziumchenko NE, Anisimov AP (2001) [Evolutionary regularities of the appearance of polyploidy in salivary glands of gastropod mollusks. III. A subclass of Pectinibranchia : Orders Discopoda, Echinospirida, Aspidophora and Entomostoma (Mesogastropoda)]. Tsitologiia 43 :553-560


By means of histological methods and DNA cytophotometry, a study was made of the salivary glands of 16 species of gastropod molluscs belonging to the subclass Pectinibranchia and making a group of Mesogastropoda. Four cell types of salivary glands were distinguished : granular cells (with glycoproteid granular inclusions), mucocytes-I (with sulfatic acid mucopolysaccharides), mucocytes-II (with neutral and acid polysaccharides and proteins), and also the epithelial ciliated cells and mucous duct cells. Data of experiments on starvation and synchronous feeding of molluscs have testified that all described cell types are independent. In some species differentiation on protein and mucous departments within the glandular epithelium was shown. In some marine representatives of the orders Discopoda and Aspidophora polyploid cells with the ploidy levels from 8c to 32c were revealed along with diploid cells. The ecological and phylogenetic regularities of somatic polyploidy manifestation in Mesogastropoda are discussed.

Zubkov MV, Fuchs BM, Burkill PH, Amann R (2001) Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea. Appl Environ Microbiol 67 :5210-5218


A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [(35)S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of alpha-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the alpha-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of gamma-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production : different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.

Zubkov MV, Zollner E, Jurgens K (2001) Digestion of bacterial macromolecules by a mixotrophic flagellate, Ochromonas sp., compared with that by two heterotrophic flagellates, Spumella pudica and Bodo saltans. European Journal of Protistology 37 :155-166

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Digestion of bacterial biomass by three species of phagotrophic flagellates was studied using radioactive tracer techniques and short-term feeding experiments. Macromolecules of two different bacterial strains and natural limnic bacterioplankton were pulse-chase-labelled with one of the following precursors H-3-thymidine, S-35-/C-14-methionine or C-14-leucine, before bacteria were fed to flagellates and radioactive labels were traced into flagellate macromolecules. The concentrations of prey and predators were monitored by flow cytometry. The aim of the work was to compare efficiencies of bacterial macromolecule accumulation by mixotrophic (Ochromonas) and heterotrophic (Spumella and Bodo) flagellates. We observed that flagellate accumulation efficiency of bacterial macromolecules labelled with thymidine (mean 15-30%, depending on flagellate species) was lower than of bacterial macromolecules labelled with amino acids (mean 26-68%). Heterotrophic flagellate species had similar accumulation efficiencies of bacterial molecules, when either leucine (26-42%) or methionine (31-41%) was used as a tracer. In contrast the mixotrophic flagellate accumulated significantly more residues of labelled methionine (68%) than of labelled leucine (54%). Methionine seems to be accumulated as an intact molecule and possibly Ochromonas preferentially accumulated methionine as an additional source of reduced sulphur. Protozoan accumulation efficiencies did not differ significantly whether the pulse-labelled bacterial prey were from growing or long-term starvation cultures. Our results suggest that labelled amino acids are more appropriate than labelled thymidine for studying transfer of bacterial biomass within food webs.