Abe F (1998) Hydrostatic pressure enhances vital staining with carboxyfluorescein or carboxydichlorofluorescein in Saccharomyces cerevisiae : efficient detection of labeled yeasts by flow cytometry. Appl Environ Microbiol 64 :1139-1142
The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required D-glucose and was markedly inhibited by 2-deoxy-D-glucose, suggesting that glucose metabolism has a role in the process.
Ashton-Alcox KA, Ford SE (1998) Variability in molluscan hemocytes : a flow cytometric study. Tissue Cell 30 :195-204
Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to : (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.
Barer MR, Kaprelyants AS, Weichart DH, Harwood CR, Kell DB (1998) Microbial stress and culturability : conceptual and operational domains. Microbiology-Uk 144 :2009-2010
Bianchi M (1998) New approaches to study microbial networks. Annales De Limnologie-International Journal of Limnology 34 :465-473
Aquatic ecosystems are characterised by physical and chemical fluctuations and gradients as well as by the resulting transportation’s of organisms, modifications of communities and exchanges between individuals. The microbial networks, from the phytoplankton to the bacteria and protozoa, all in various states of activity, are a major component for the functioning of the pelagic ecosystems. The measurement of in situ numbers and activities of the micro-organisms is a permanent challenge for the microbiologist. Recent advances in techniques and technologies has provided new concepts to study micro-organisms in natural aquatic environments. They concern i) the improvement of counts of viable bacteria, ii) the combination of cytometry techniques (image analysis and/or flux cytometry) with fluorescent markers (fluorochromes, fluorescent genetic probes) to determine species or metabolic function, iii) the use of radioactive compounds combined with techniques of fine chemistry like GC, HPLC, mass spectrometry to describe metabolic rates, interaction with the chemical environment, iv) the assessment of bacterial response to external stresses at the molecular level.
Binder BJ, Liu YC (1998) Growth rate regulation of rRNA content of a marine synechococcus (Cyanobacterium) strain. Appl Environ Microbiol 64 :3346-3351
The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells (&mgr; = 0.15 day-1). The relationship between growth rate and cellular rRNA content comprised three phases : (i) at low growth rates (< approximately 0.7 day-1), rRNA cell-1 remained approximately constant ; (ii) at intermediate rates ( approximately 0. 7 - 1.6 day-1), rRNA cell-1 increased proportionally with growth rate ; and (iii) at the highest, light-saturated rates (> approximately 1.6 day-1), rRNA cell-1 dropped abruptly. Total cellular RNA (as measured with the nucleic acid stain SYBR Green II) was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration (amount of rRNA per cubic micrometer) were related to growth rate in a manner similar to rRNA cell-1, although the overall magnitude of change in both cases was reduced. These patterns are hypothesized to reflect an approximately linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.
Boddy L, Morris CW (1998) Approaching automated quantification of marine phytoplankton populations by neural network analysis of flow cytometry data. Ocean Community Conference’98 : Celebrating 1998 International Year of the Ocean, Proceedings Vols 1 and 2 :440-444 1196
It is essential to be able to determine composition and size of phytoplankton communities as they are key components of marine ecosystems. Analytical flow cytometry (AFC) can provide ’fingerprints’ (in terms of light scatter and fluorescence) of these microalgal cells, at rates of 10(3) cells sec(-1). To make full use of this technology a rapid (near real time) data analysis system is required. Artificial neural networks (ANNs) provide a solution. The approach is illustrated using AFC data on ii important marine phytoplankton taxa. Supervised and unsupervised ANNs are described, and ways of overcoming problems in obtaining training data, overlapping character distributions, unbounded data sets and scaling up are discussed.
Button DK, Robertson BR, Lepp PW, Schmidt TM (1998) A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Applied and Environmental Microbiology 64 :4467-4476
Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible ; it was named Cycloclasticus oligotrophus. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant K-A for toluene was low at 1.3 mu g/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme ; K-A increased with increasing specific affinities. Specific affinities, a(s)(o), were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a(s)(o) and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.
Castano A, Carbonell G, Carballo M, Fernandez C, Boleas S, Tarazona JV (1998) Sublethal effects of repeated intraperitoneal cadmium injections on rainbow trout (Oncorhynchus mykiss). Ecotoxicol Environ Saf 41 :29-35
Acute and chronic effects of cadmium have been widely described for different aquatic organisms and exposure routes. However, there is clearly a lack of information on the potential of cadmium to cause genotoxic effects. This work presents genotoxic and nongenotoxic parameters analyzed in cadmium-exposed rainbow trout. The assessment was performed for sublethal levels after long-term exposure using six intraperitoneal injections of 0.5 mg/kg (Day 1), 1 mg/kg (Days 3, 7 and 11), and 2 mg/kg (Days 15 and 19) to allow precise estimation of the dose. Cadmium accumulation in target tissues, essential metal mobilization by cadmium at the subcellular and tissue levels, and induction of metallothioneins were selected as exposure and effect parameters. Induction of micronuclei and variation in DNA content (expressed as variation coefficient in the G1 phase of the cell cycle) in blood cells, determined by flow cytometry, were selected as biomarkers for genotoxic effects. Cadmium accumulation, induction of metallothioneins, and mobilization of essential metals at the subcellular level were observed in different organs in response to cadmium exposure. The highest metallothionein induction was observed in liver, reaching 270+/-90 nmol/g wet tissue in treated fish versus 2.68+/-1.1 nmol/g wet tissue in controls. The highest cadmium accumulation was also observed in the liver (27.8+/-9.5 microgram Cd/g wet wt in treated animals versus 1.0+/-1.7 in the control group). However, no genotoxic effects were observed in blood cells. The frequency of micronuclei was 0.012+/-0.008 for the control group and 0.013+/-0.021 for treated animals. The variation coefficient of G1-phase nuclei was 3.61+/-0.66 and 3.22+/-0.29 for control and cadmium-exposed groups, respectively. Thus, it is concluded that under the experimental conditions employed here, treatment of rainbow trout with cadmium chloride at doses that produce significant toxicological alterations at the tissue and subcellular levels does not provoke observable alterations in the genotoxic parameters considered in this study.
Courties C, Perasso R, Chretiennot-Dinet MJ, Gouy M, Guillou L, Troussellier M (1998) Phylogenetic analysis and genome size of Ostreococcus tauri (Chlorophyta, Prasinophyceae). Journal of Phycology 34 :844-849
Ostreococcus tauri Courties et Chretiennot-Dinet is the smallest described autotrophic eukaryote dominating the phytoplanktonic assemblage of the marine Mediterranean Thau lagoon (France). Its taxonomic position was partly elucidated from ultrastructure and high-pressure liquid chromatography (HLPC) pigment analysis. The sequence analysis of the 18S rDNA gene of O. tauri measured here is available in EMBL Nucleotide Sequence Database (accession number : Y15814) and allowed to clarify its phylogenetic position. O. tauri belongs to the Prasinophyceae and appears very close to Mantoniella, a typical scaly Prasinophyceae, morphologically very different from the naked and coccoid Ostreococcus. An electrophoretic analysis of O. tauri shows that the nucleus contains 10.20 mbp. This small genome fragmented into 14 chromosomes ranging in size from 300 to 1500 kbp, confirms the minimalist characteristics of Ostreococcus tauri.
De Guise S, Erickson K, Blanchard M, Dimolfetto L, Lepper H, Wang J, Stott JL, Ferrick DA (1998) Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R. Immunology 94 :207-212
As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.
Downes MT, Hall JA (1998) A sensitive fluorometric technique for the measurement of phycobilin pigments and its application to the study of marine and freshwater picophytoplankton in oligotrophic environments. Journal of Applied Phycology 10 :357-363
A sensitive and specific technique is described for the estimation of playcobiliprotein in freshwater and marine picophytoplankton. The method uses fluorescent properties to detect phycoerythrin concentrations as low as 40 ng L-1 from a 1 L water sample and is capable of distinguishing between R-phycoerythrin, C-phycocyanin and C-phycoerythrin. The application of the method to the study of natural picophytoplankton populations in marine and freshwater environments is described. Nitrate concentrations appear to influence picophytoplankton cellular C-phycoerythrin concentrations in surface waters and increasing cellular C-phycoerythrin fluorescence with water depth suggests that this pigment plays a role as a photosynthetic accessory pigment.
Fuchs BM, Wallner G, Beisker W, Schwippl I, Ludwig W, Amann R (1998) Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 64 :4973-4982
In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083(T). Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. 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 with Eco1482, respectively) was as follows : I, 4% ; II, 14% ; III, 21% ; IV, 29%, V, 19% ; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.
Gonzalez-Gil S, Keafer BA, Jovine RVM, Aguilera A, Lu SH, Anderson DM (1998) Detection and quantification of alkaline phosphatase in single cells of phosphorus-starved marine phytoplankton. Marine Ecology-Progress Series 164 :21-35
Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P compounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on individual species. A new insoluble fluorogenic substrate for AP, termed ELF (Enzyme-Labeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme activity and thus has the capability to determine the P status of individual cells. In this study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-O-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Amphidinium sp. and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 mu M (i.e, f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate reader (Cytofluor(TM)). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipitate. This signal was always observed for P-starved Amphidinium sp, and I. galbana cells, but was seen in some A. fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A, fundyense, cells grown in low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable analysis using the soluble substrates MFP, FDP, and Attophos(TM) on the Cytofluor showed little activity for A. fundyense, but high fluorescence for P-starved Amphidinium sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or now cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters.
Jacquet S, Lennon JF, Marie D, Vaulot D (1998) Picoplankton population dynamics in coastal waters of the northwestern Mediterranean Sea. Limnology and Oceanography 43 :1916-1931
High-frequency sampling of surface picoplanktonic populations was performed in Villefranche Bay (north-western Mediterranean Sea) during the first week of July 1996. The evolution of abundance and cell parameters were monitored once per hour by flow cytometry for these populations : Synechococcus cyanobacteria, photosynthetic picoeukaryotes, and heterotrophic bacteria plus Prochlorococcus. Some parameters, such as the right-angle light scatter of Synechococcus and picoeukaryotes, or the red chlorophyll fluorescence of picoeukaryotes, exhibited a very clear 24-h diel periodicity. For other parameters, such as Synechococcus red chlorophyll fluorescence, it was necessary to perform a Fourier analysis to establish a major 24-h period unambiguously. This analysis also revealed that some other parameters, however, such as the cell concentration, right-angle light scatter, and red chlorophyll fluorescence of picoeukaryotes, or the red chlorophyll and orange phycoerythrin fluorescence of Synechococcus, had a period of 17-18 h, corresponding to the inertial frequency at this latitude. The cell cycle of Synechococcus, sampled twice per hour, was synchronized with the daily light cycle, allowing an estimate of their growth rate, which averaged 0.95 d(-1) (SD = 0.26, n = 7). Its large day-to-day variability was related to the duration of the interval between the maxima of S and G2 phases that ranged from 2 to 3.5 hours. Generally, the division rate was depressed on sunny days. The loss rate of Synechococcus was in general lower than the division rate and appeared to follow the evolution of the latter with a 1-d lag, as if grazers or viruses adapted very rapidly to changes in division rates. Over the period of study, concentrations of Synechococcus and other bacteria were significantly correlated (r(2) = 0.60, p < 0.01, n = 340), suggesting the possibility of a common controlling factor for these populations, e.g., phosphorus or grazing.
Kapraun DF, Buratti JR (1998) Evolution of genome size in the Dasycladales (Chlorophyta) as determined by DAPI cytophotometry. Phycologia 37 :176-183
Microspectrophotometry with the DNA-localizing fluorochrome 3’,6-diamidino-2-phenylindole (DAPI) was used to estimate nuclear genome sizes in 12 species representing two families of the Dasycladales. Estimated 2C DNA contents in these taxa range from 0.7-1.4 pg in the family Dasycladaceae and from 0.9-2.4 pg in the family Acetabulariaceae. In both families, species exhibiting characteristics considered to be primitive appear to have 2C DNA contents that approximate 50% of values found in species exhibiting characteristics considered to be more advanced. Results suggest that polyploidy events have accompanied evolution in the order but apparently have been infrequent and conservatively preserved, leading to a narrow range of relatively small genome sizes. Limitations on genome size increase are discussed in terms of constraints imposed by a prolonged uninucleate stage. Interspecific comparisons of genome size and gametangium volume suggest an inverse relationship. Gametangium volume appears to have phylogentic significance, in that taxa with more advanced characteristics generally have smaller gametangia.
Lebaron P, Parthuisot N, Catala P (1998) Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl Environ Microbiol 64 :1725-1730
Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4’,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.
Lopez-Amoros R, Comas J, Garcia MT, Vives-Rego J (1998) Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation. Fems Microbiology Ecology 27 :33-42
Viable, total and metabolically active bacteria were determined during linear alkylbenzene sulfonate degradation in coastal seawater. Viable bacteria were estimated by plate counts on marine agar media while the total and metabolically active bacteria were determined with the nucleic acid stain SYTO-13 and the tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride, respectively, in double stain procedures analyzed by flow cytometry. The double stain SYTO-13/5-cyano-2,3-ditolyl tetrazolium chloride is a rapid and simple method that discriminates bacterioplankton populations according to nucleic acid content and formazan formation. Linear alkylbenzene sulfonate degradation was monitored by high-performance liquid chromatography analysis. Bacterioplankton degraded linear alkylbenzene sulfonate by growing to communities with a high percentage of viable and metabolically active bacteria. The bacteria produced were rapidly grazed by protozoa ; however, the grazing took place mostly on metabolically active cells, which were larger than the rest of the population. (C) 1998 Published by Elsevier Science B.V. All rights reserved.
Mackey DJ, Higgins HW, Mackey MD, Holdsworth D (1998) Algal class abundances in the western equatorial Pacific : Estimation from HPLC measurements of chloroplast pigments using CHEMTAX. Deep-Sea Research Part I-Oceanographic Research Papers 45 :1441-1468
Samples for the analysis of phytoplankton photosynthetic pigments were collected from the equatorial Pacific (5 degrees N to 15 degrees S along 155 degrees E) in October 1990 as part of the Australian contribution to the JGOFS program. Chlorophyll and carotenoid pigments were measured by HPLC using a diode-array detector. A PC-based computer program was used to optimise the pigment ratios and to estimate the contributions of 10 algal classes to the total chlorophyll a concentration at each location and in 7 separate depth bands. For the pigments that occur in more than one algal class, the pigment : chlorophyll a ratios for 19’-butanoyloxyfucoxanthin and 19’-hexanoyloxyfucoxanthin (chrysophytes and haptophytes), neoxanthin (prasinophytes, euglenophytes and chlorophytes) and chlorophyll b (prasinophytes, euglenophytes, prochlorophytes and chlorophytes) increase with depth, while those of violaxanthin (prasinophytes and chlorophytes), diadinoxanthin (dinoflagellates, chrysophytes, haptophytes, euglenophytes and diatoms), lutein (prasinophytes and chlorophytes) and, zeaxanthin (prasinophytes, cyanobacteria, prochlorophytes and chlorophytes) decrease with depth. Peridinin : chlorophyll a increases with depth in dinoflagellates, while alloxanthin : chlorophyll a decreases with depth in cryptomonads. The only pigment ratio that does not change consistently with depth is that of fucoxanthin, which increases with depth in chrysophytes and haptophytes but decreases in diatoms. Based on their contribution to the total chlorophyll a, cyanobacteria (Synechococcus) were dominant in the nutrient depleted surface waters, haptophytes were dominant at mid depth (70 m), and prochlorophytes were dominant at depths of 100-125 m. These three algal classes were by far the most important, and each contributed up to 30-40% of the total chlorophyll a at some depth within the water column. Chlorophytes and chrysophytes contributed up to a maximum of about 12% of the total chlorophyll a, while cryptophytes, diatoms, dinoflagellates, prasinophytes and (possibly) euglenophytes generally contributed up to 4-8% of the chlorophyll a. (C) 1998 Elsevier Science Ltd. All rights reserved.
Maurin-Defossez C, Le Gal Y (1998) Diel periodicity of glutamine synthetase activity during the cell cycle of Emiliania huxleyi. Plant Physiology and Biochemistry 36 :233-236
Light-dark regulation of glutamine synthetase (GS ; EC 184.108.40.206) was investigated by following the development of activity and the time-course of the cell-cycle of the marine coccolithophorid, Emiliania huxleyi, under a 16-h photoperiod in synchronous cultures. GS activity showed marked diel periodicity by exhibiting a maximum value during the dark period and a minimum during the light. Our results suggest that regulation of GS activity during the light-dark cycle probably does not operate only through activation/deactivation mechanisms. Cell division took place from the end of the dark to the first hours of the light period. The succession of the cell-cycle events was analysed by flow cytometry. Variations in GS activity appeared to match the demands of the cell for glutamine, increasing during the G(1) phase in the light, and remaining at a high level during DNA synthesis in the dark. (C) Elsevier, Paris.
Minor EC, Eglinton TI, Olson R, Boon JJ (1998) The compositional heterogeneity of particulate organic matter from the surface ocean : an investigation using flow cytometry and DT-MS. Organic Geochemistry 29 :1561-+
In this study the morphological and chemical heterogeneity of particulate organic matter (POM) in the surface ocean was investigated by the application of Bow cytometry, direct temperature-resolved mass spectrometry (DT-MS), and multivariate statistics. In order to obtain a diverse collection of surface-ocean POM samples, POM was collected from a station in Great Harbor, Woods Hole, Mh, U.S.A. ton a monthly basis) and on transects across the Mid-Atlantic Eight tin March 1996). Small particle (<53 mu m) POM was separated from large-particle (>53 mu m) POM by filtration through a nylon screen. Aliquots of the small-particle POM were then further separated. via Bow cytometric sorting, into phytoplankton and detrital pools. Broad-band molecular-level characteristics of these POM subclasses were obtained through DT-MS (16 eV, EI+) and were explored via principal component and discriminant analyses. For both the Woods Hole (WHTS) and the Mid-Atlantic Eight (MAB) data sets, the statistical treatments separated POM samples primarily according to their class and subclass distinctions ; therefore, this paper focuses on the molecular-level differences among large-particle and small-particle POM, phytoplankton and detritus. Small-particle POM is found to be more enriched in protein, phytosterols, diglycerides and chlorophyll than large-particle POM, which is more enriched in pentose polysaccharide fragments, C-16:0 fatty acid, and cholesterol. At the WHTS site, large-particle POM is also enriched in chitin and appears to contain a significant grazer biomass component. In the MAB, the corresponding POM fraction appears more phytodetrital. Phytoplankton particles are enriched in protein, chlorophyll and lipids relative to detritus. Detritus is enriched in selected polysaccharides and appears to be composed of phytodetritus and/or fecal material, (C) 1998 Elsevier Science Ltd. All rights reserved.
Moreira-Turcq PF, Martin JM (1998) Characterisation of fine particles by flow cytometry in estuarine and coastal Arctic waters. Journal of Sea Research 39 :217-226
The chemical and biological nature of suspended pico- (<2 mu m) and nano- (2-10 mu m) particles was studied by flow cytometry in the Lena River delta and Laptev Sea, Russia, during September 1991. Forward and wide-angle light scatter, natural fluorescence of phytoplankton, and induced fluorescence of organic molecules were used to characterise natural suspended particles. Organic and inorganic particles were identified by staining with specific fluorochromes : FITC for proteins, Con A-FITC for glucose/mannose, and PNA for galactose. Living and nonliving organic particles were distinguished by analysing simultaneously natural red fluorescence (chlorophyll) and organic staining. The upper Lena River and its delta were characterised by a high concentration of total particles (18.5 +/- 4.9 x 10(5) per cm(3)), mostly inorganic (13.6 +/- 5.4 x 10(5) per cm(3)), in the coastal and open waters of the Laptev Sea, organic particles dominated. Generally, the most important fraction of small organic particles were nonliving (organic detritus, TEP, and organic coatings) characterised by the presence of proteins and polysaccharides. The phytoplanktonic cells were characterised by a high fraction of picoplankton (1000-50 000 cells per cm(3)) dominated by Synechococcus sp. and small picoeukaryotes. (C) 1998 Elsevier Science B.V. All rights reserved.
O’Dowd AM, Ellis AE, Secombes CJ (1998) Binding of immune complexes to Atlantic salmon peripheral blood leucocytes. Dev Comp Immunol 22 :439-448
The ability of peripheral blood leucocytes (PBL) of Atlantic salmon to bind immune complexes in an antibody-dependent fashion was investigated. Immune complexes were labelled with fluorescein isothiocyanate and binding of these complexes to isolated PBL was determined by flow cytometry. The data show that a high proportion (up to 65%) of PBL were capable of binding immune complexes, and this binding did not occur when immune serum was replaced with normal serum. The presence of fresh normal serum inhibited or abrogated immune complex-binding of PBL. This is the first report of high levels of immune complex receptors on leucocytes in fish, and the dependence of complex binding on the presence of antibody suggests that these receptors may be similar to Fc receptors which are widely distributed on immunocytes of mammals.
Ottaviani E, Franchini A, Barbieri D, Kletsas D (1998) Comparative and morphofunctional studies on Mytilus galloprovincialis hemocytes : presence of two aging-related hemocyte stages. Italian Journal of Zoology 65 :349-354
Morphological and functional studies were performed on hemocytes from young and adult Mytilus galloprovincialis. The results obtained suggest that only one cell type in two different stages, young or old, is present, consistently with the Mix’s one-cell-type model. However, both young and old hemocytes appear capable of carrying out similar functions and express common signal molecules ; thus, their differences in cytology and number appear to be due to cellular aging.
Quang PX, Button DK (1998) Use of species distribution data in the determination of bacterial viability by extinction culture of aquatic bacteria. Journal of Microbiological Methods 33 :203-210
We show that bacterial viability can be calculated from the number of species arising in cultures comprised of seawater diluted to near extinction of the propagating organisms. Natural conditions are simulated by partitioning the culture against illuminated raw seawater to maintain a source of nutrients and a sink for waste products, and predators are eliminated by dilution to a statistical absence. Mean values for viability can be corrected for contaminants that may enter the culture by computing their probable entrance frequency as well. Viability determinations were found to be mildly sensitive to the number of species estimated to be present in the original sample. This novel procedure provides a significant improvement in the cultivation success of typical marine bacteria, and the formulations presented here lay the foundation for the quantitative analysis of the population structure. (C) 1998 Elsevier Science B.V. All rights reserved.
Rappe MS, Suzuki MT, Vergin KL, Giovannoni SJ (1998) Phylogenetic diversity of ultraplankton plastid small-subunit rRNA genes recovered in environmental nucleic acid samples from the Pacific and Atlantic coasts of the United States. Applied and Environmental Microbiology 64 :294-303
The scope of marine phytoplankton diversity is uncertain in many respects because, like bacteria, these organisms sometimes lack defining morphological characteristics and can be a challenge to grow in culture. Here, we report the recovery of phylogenetically diverse plastid small-subunit (SSU) rRNA gene (rDNA) clones from natural plankton populations collected in the Pacific Ocean off the mouth of Yaquina Bay, Oreg. (OCS clones), and from the eastern continental shelf of the United States off Cape Hatteras, N.C. (OM clones), SSU rRNA gene clone libraries were prepared by amplifying rDNAs from nucleic acids isolated from plankton samples and cloning them into plasmid vectors, The PCR primers used for amplification reactions were designed to be specific for bacterial SSU rRNA genes ; however, plastid genes have a common phylogenetic origin with bacteria and were common in both SSU rRNA gene clone libraries, A combination of restriction fragment length polymorphism analyses, nucleic acid sequencing, and taxon-specific oligonucleotide probe hybridizations revealed that 54 of the 116 OCS gene clones were of plastid origin, Collectively, clones from the OCS and OM libraries formed at least eight unique lineages within the plastid radiation, including gene lineages related to the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae, Chrysophyceae, and Prasinophyceae ; for a number of unique clones, no close phylogenetic neighbors could be identified with confidence, Only a group of two OCS rRNA gene clones showed close identity to the plastid SSU rRNA gene sequence of a cultured organism [Emiliania huxleyi (Lohmann) Hay and Mohler ; 99.8% similar]. The remaining clones could not be identified to the genus or species level, Although cryptic species are not as prevalent among phytoplankton as they are among their bacterial counterparts, this genetic survey nonetheless uncovered significant new information about phytoplankton diversity.
Robertson BR, Button DK, Koch AL (1998) Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl Environ Microbiol 64 :3900-3909
The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4’, 6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 &mgr;g/liter. The lowest value for mean dry mass, 0.027 +/- 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.
Sharma RV, Edwards RT, Beckett R (1998) Analysis of bacteria in aquatic environments using sedimentation field-flow fractionation : (II) Physical characterization of cells. Water Research 32 :1508-1514
This paper describes the application of sedimentation field-flow fractionation (SdFFF) for the physical characterization of bacteria cells found in natural aquatic environments. This technique separates bacteria on the basis of the buoyant mass of the cells and fractions can be collected for determination of cell numbers and volume. This enables the cell density and size distribution of the sample to be calculated. SdFFF is also capable of generating very detailed cell size and specific gravity distributions which may be useful data in assessing the bacterial growth status of natural waters. Data for samples from seawater, two lakes and a sewer drain are given. The specific gravity of cells varies both within and between samples and falls in the range 1.03-1.2 g cm(-3). This contrasts with previous work which shows little variation within a given cultured bacteria sample and indicates that natural populations contain a range of different bacteria types. (C) 1998 Elsevier Science Ltd. All rights reserved.
Sieracki CK, Sieracki ME, Yentsch CS (1998) An imaging-in-flow system for automated analysis of marine microplankton. Marine Ecology-Progress Series 168 :285-296
Present automated systems for counting and measuring marine plankton include flow cytometers and in situ plankton video recorders. Neither of these approaches are optimal for the microplankton cells which range in size from 20 to 200 mu m and can be fewer than 10(4) l(-1). We describe here an instrument designed for rapid counting, imaging and measuring of individual cells and particles in the microplankton size range from cultures and natural populations. It uses a unique optical element to extend the depth of focus of the imaging lens, allowing a sample stream flow rate of 1 ml min(-1) The instrument stores a digital image of each particle along with real time fluorescence and size measurements. An interactive cytogram links a dot-plot of the size and fluorescence data to the stored cell images, allowing rapid characterization of populations. We have tested the system on live phytoplankton cultures and bead standards, proving the system counting and sizing accuracy and precision. The system provides images and size distributions for cultures or natural marine samples. It has been used successfully at sea to continuously monitor particles while underway. It may prove useful in studies of plankton community structure, ocean optics and monitoring for harmful algal species.
Smith EM (1998) Coherence of microbial respiration rate and cell-specific bacterial activity in a coastal planktonic community. Aquatic Microbial Ecology 16 :27-35
The fluorogenic tetrazolium dye 5-cyano-2, 3 ditolyl tetrazolium chloride (CTC) has been increasingly used as a measure of cell-specific metabolic activity in bacteria, in that it acts as an electron acceptor in the electron transport system (ETS) of bacterial cells. As such, it is taken to be a direct measure of abundance of those bacterial cells that are actively engaged in respiration. Although it would, thus, be anticipated that microbial respiration should he strongly correlated to the number of CTC-active cells, such a relationship has yet to be demonstrated for natural bacterioplankton communities. CTC was used in situ to assay cell-specific respiratory activity within the pelagic community of Chesapeake Bay. Over the course of sampling, the observed variation in CTC-active cell abundance was 25-fold, substantially greater than the 6-fold variation in total cell abundance (estimated by DAPI staining). As a result, the proportion of CTC-active cells ranged from 3.5 to 47.4 % of the total bacterial population, with this proportion varying seasonally as well as spatially. Both abundance and proportion of CTC-active cells were highly correlated with respiration rates within the microplankton community (<3 mu m size fraction), explaining 80%, or more, of the variations in these rates. Although respiration rates and total bacterial abundance also tended to covary seasonally, the relationship was not as strong, and large spatial differences in respiration along the Bay could not be explained by total abundance alone. This suggests that the large spatial/temporal variations previously observed in respiration rates are the result of changes in the number of active bacteria, rather than total bacterial abundance. Results of the present study thus encourage the view that CTC provides an ecologically meaningful measure of active bacterial abundance in aquatic systems, and that CTC-active bacteria are likely responsible for the hulk of bacterial community metabolic activity.
Sogawa K, Kodama E, Matsuda M, Shigeta S, Okutani K (1998) Marine microalgal polysaccharide induces apoptosis in human lymphoid cells. Journal of Marine Biotechnology 6 :35-38
An extracellular polysaccharide from the marine microalga dinoflagellate Gymnodinium sp, A3, which was a D-galactan sulfate associated with L(+)-lactic acid, showed cytotoxicities to various human lymphoid cells, especially to MT-4 cells (CC50 2.67 mu g/ml). Close observations of morphological change, flow cytometry, and in situ end-labeling of fragmentated DNA revealed the mechanism of cytotoxicity of this polymer to be based on the induction of apoptosis.
Troussellier M, Bonnefont JL, Courties C, Derrien A, Dupray E, Gauthier M, Gourmelon M, Joux F, Lebaron P, Martin Y, Pommepuy M (1998) Responses of enteric bacteria to environmental stresses in seawater. Oceanologica Acta 21 :965-981
The effects of different environmental factors (nutrient deprivation, hyperosmotic shock, exposure to light) on enteric bacteria which have been transferred into the marine environment, have been studied experimentally (microcosms) by considering demographic, physiological and genetic responses in Escherichia coli or Salmonella typhimurium populations. Short-term experiments (less than or equal to 48 h) showed that nutrient deprivation induced limited changes in measured bacteriological variables, but when combined with hyperosmotic shock, it results in an energy charge decrease and inactivation of membrane transport. Light exposure mainly affects the colony-forming capacity of bacterial populations. Combining different stress factors confirmed the rapid appearance of a viable, but nonculturable state (VBNC) in populations of E. coli and S. typhimurium. It has been shown that cellular forms other than those previously described in the literature can be generated following incubation in seawater. It was also established that pre-adaptation phenomena may occur, leading to better survival (e.g. pre-incubation in seawater in darkness enhanced survival under light exposure). An explanation concerning these phenomena can be found by looking at the rpoS gene which controls the expression of numerous genes and can trigger a general anti-stress response under different adverse conditions. Although the results provide better comprehension of the fate of enteric bacteria in the marine environment, they also raise numerous questions related to fundamental and applied problems, given in the conclusion of this paper. (C) Elsevier, Paris.
van Hannen EJ, van Agterveld MP, Gons HJ, Laanbroek HJ (1998) Revealing genetic diversity of eukaryotic microorganisms in aquatic environments by denaturing gradient gel electrophoresis. Journal of Phycology 34 :206-213
A new Eucarya-specific 18S rDNA primer set was constructed and tested using denaturing gradient gel electrophoresis to analyze the genetic diversity of eukaryotic microorganisms in aquatic environments. All eukaryal lines of descent exhibited four or fewer nucleotide mismatches in the forward primer sequence, except for the microspora line of descent. The reverse primer annealed to a more conserved region with fewer than two nucleotide mismatches. Genomic DNA from test organisms with different numbers of nucleotide mismatches were amplified to test primer specificity. Relatively low annealing temperatures allowed the amplification of sequences with up to four nucleotide mismatches while still maintaining specificity for the eukaryal domain. Denaturing gradient gel electrophoresis was used to separate similarly sized PCR products of environmental samples, and the obtained banding patterns were converted to a binary format for statistical comparisons. Cluster analysis of these patterns showed similar results to a cluster analysis based on environmental variables. This approach provides an analytical tool to study the population structure and molecular ecology of eukaryotic microbial communities inhabiting aquatic environments.
van Leeuwe MA, Timmermans KR, Witte HJ, Kraay GW, Veldhuis MJW, de Baar HJW (1998) Effects of iron stress on chromatic adaptation by natural phytoplankton communities in the Southern Ocean. Marine Ecology-Progress Series 166 :43-52
Effects of iron stress on chromatic adaptation were studied in natural phytoplankton communities collected in the Pacific region of the Southern Ocean. Iron enrichment experiments (48 to 72 h) were performed, incubating plankton communities under white, green and blue light respectively, with and without addition of 2 nM Fe. Pigment ratios were affected by iron addition only to a minor extent. The pigment composition as dictated by the light conditions was similar for both the iron-enriched and the unamended bottles. Upon iron addition, phytoplankton auto-fluorescence, as estimated by flow cytometry, decreased markedly, indicating iron stress of the endemic phytoplankton community. It was concluded that iron did not control chromatic adaptation via the pigment composition, but exerted a clear effect on the efficiency of electron transfer.
Williams SC, Hong Y, Danavall DCA, Howard-Jones MH, Gibson D, Frischer ME, Verity PG (1998) Distinguishing between living and nonliving bacteria : Evaluation of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples. Journal of Microbiological Methods 32 :225-236
Recent studies have suggested that a large fraction of marine bacterioplankton are either dead or moribund and, therefore, new methods are required to distinguish bacteria of different physiological states. A method is described which uses the general cell stain 4’6’-diamidino-2-phenylindole (DAPI), the vital stain propidium iodide (PI), and 16S rRNA-targeted oligonucleotide probes. to quantitatively identify cells with compromised membranes, and those cells containing sufficient rRNA to be considered metabolically active. Validation and optimization of this method was conducted using cultured bacteria. Optimal PI staining was achieved after cells were washed in 10 mM MgSO4 (pH=6.5) and stained with PI (5 mu g/ml) for 30 min. Staining of cells with PI appeared to be independent of growth phase and cells could be stored in 25% (v/v) glycerol for at least one month at -20 degrees C without changes ir. staining status. Staining of heat-killed cells indicated that PI stained only dead cells. Comparison of PI staining properties and hybridization with 16S rRNA-targeted oligonucleotide probes indicated that there was a strong inverse correlation between hybridization of cells with 16S rRNA-targeted oligonucleotide probes and cells stained by PI. Evidence indicates that this viral stain and probe (VSP) technique differentiates between (1) cells that are dead, (2) cells that are dead but were recently active (<36 h), (3) cells that are living and (4) cells that are inactive but not dead. The VSP protocol comprises a powerful tool to investigate the relative importance of these cell types in situ, and how they change in response to environmental factors. (C) 1998 Published by Elsevier Science B.V.
Wood AM, Phinney DA, Yentsch CS (1998) Water column transparency and the distribution of spectrally distinct forms of phycoerythrin-containing organisms. Marine Ecology-Progress Series 162 :25-31
Predominance of Type I (phycourobilin-containing) and Type II (phycourobilin-lacking) phycoerythrins (PE) was examined using scanning fluorescence spectroscopy at 176 stations in the northwestern Atlantic off the northeast coast of the United States. Simultaneous optical measurements were made at 75 stations, permitting an analysis of the distribution of spectral types of PE-containing organisms based on geographic position of the stations and on the relative penetration of blue and green wavelengths of light. Stations dominated by Type I PE occurred almost exclusively in very transparent water with high transmissivity for blue light [domnwelling attenuation coefficient ; K-d(440) < 0.12] and relatively low attenuation of blue light relative to green light. This pattern was reversed for Type II PE, which dominated in less transparent waters with relatively high attenuation of blue light relative to green light. Type II PE tended to dominate on the continental shelf and slope, and Type I PE tended to dominate in the Sargasso Sea. Regardless of geographic location, there was a transition from dominance by Type I PE to Type II PE as the ratio K-d(440)/K-d(550) exceeded 1.25. Our data suggest that optical parameters are important niche dimensions for marine Synechococcus and that nearshore waters may be classified optically by phycoerythrin characterization.
Zubkov MV, Sleigh MA, Tarran GA, Burkill PH, Leakey RJG (1998) Picoplanktonic community structure on an Atlantic transect from 50 degrees N to 50 degrees S. Deep-Sea Research Part I-Oceanographic Research Papers 45 :1339-1355
Plankton samples were collected from 10 depths at 25 stations spaced at intervals of about 4 degrees of latitude along a transect from the British Isles to the Falkland Islands. Four categories of picoplankton were discriminated : Synechococcus spp., Prochlorococcus spp., eukaryotic picophytoplankton and heterotrophic bacteria. The populations in each category in the samples were counted by flow cytometry and the mean size of bacterial cells was determined by fractionation through filters. Categories of phototrophic cells were discriminated by size and by the fluorescence of photosynthetic pigments ; samples stained with the fluorochrome TOTO were used to enumerate heterotrophic bacteria (and Prochlorococcus in surface waters where their chlorophyll content was very small). The carbon biomass concentration of each category in each sample was calculated. Prochlorococcus was present at all stations between 47 degrees N and 38 degrees S, and reached peak population densities above 200,000 cells ml(-1) in equatorial waters ; the depth occupied by these cells increased in oligotrophic waters, where they dominated picophytoplankton biomass. Synechococcus reached high concentrations in the Mauritanian upwelling region and in the frontal region near the southern end of the transect, where they represented the largest single component of picophytoplankton biomass, but was almost absent in oligotrophic regions. Picoeukaryotes were present in low numbers at all latitudes, but they are larger cells and constituted a substantial part of the total picophytoplankton biomass at most latitudes. The depth-integrated (200 m) biomass of heterotrophic bacteria was nearly as great as that of the picophytoplankton at all latitudes, because substantial numbers of cells occurred at all depths. Numbers and biomass of these bacteria were maximal in the upwelling region and high at both ends of the transect. There was a clear contrast in the composition of the picoplankton community in both the North and South Atlantic between mesotrophic waters where Synechococcus and picoeukaryotes dominated the biomass, and oligotrophic waters where the smaller total biomass was dominated by Prochlorococcus. (C) 1998 Elsevier Science Ltd. All rights reserved.