mardi 21 avril 2009
par   G. Grégori

[Anon] (2000) Aquatic flow cytometry : Achievements and prospects - Foreword. Scientia Marina 64 :119-120

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Abe F, Horikoshi K (2000) Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae. Mol Cell Biol 20 :8093-8102


Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G(1) phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not. We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa. Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15 degrees C as well as at high pressure. Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure. The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 +/- 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import. The results showing cell cycle arrest in G(1) phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin. Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function. Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.

Al-Haddad L, Morris CW, Boddy L (2000) Training radial basis function neural networks : effects of training set size and imbalanced training sets. Journal of Microbiological Methods 43 :33-44

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Obtaining training data for constructing artificial neural networks (ANNs) to identify microbiological taxa is not always easy. Often, only small data sets with different numbers of observations per taxon are available. Here, the effect of both size of the training data set and of an imbalanced number of training patterns for different taxa is investigated using radial basis function ANNs to identify up to 60 species of marine microalgae. The best networks trained to discriminate 20, 40 and 60 species respectively gave overall percentage correct identification of 92, 84 and 77%. From 100 to 200 patterns per species was sufficient in networks trained to discriminate 20, 40 or 60 species. For 40 and 60 species data sets an imbalance in the number of training patterns per species always affected training success, the greater the imbalance the greater the effect. However, this could be largely compensated for by adjusting the networks using a posteriori probabilities, estimated as network output values. (C) 2000 Elsevier Science B.V. All rights reserved.

Bernard L, Courties C, Servais P, Troussellier M, Petit M, Lebaron P (2000) Relationships among Bacterial Cell Size, Productivity, and Genetic Diversity in Aquatic Environments using Cell Sorting and Flow Cytometry. Microb Ecol 40 :148-158


The study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species.

Bernard L, Schafer H, Joux F, Courties C, Muyzer G, Lebaron P (2000) Genetic diversity of total, active and culturable marine bacteria in coastal seawater. Aquatic Microbial Ecology 23 :1-11

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The genetic diversity of marine bacteria from coastal Mediterranean water was analyzed using denaturing gradient gel electrophoresis (DGGE) and comparative sequence analysis of PCR-amplified 16S rRNA genes. The diversity of the whole bacterial assemblage was compared to the diversity of the fraction of actively respiring bacterial cells and of culturable bacteria. Culturable bacteria were isolated on agar plates using 4 different culture media, as well as in filtered autoclaved seawater following dilution to extinction. The cell fractions exhibited varied genetic diversity. High similarity between DGGE patterns obtained from the whole bacterial assemblage and those obtained from the active cell fraction (representing only 3% of total cells) indicated the simultaneous presence of both active and inactive cells within populations corresponding to numerous bacterial phylotypes defined as DGGE bands. Furthermore, an important source of genetic diversity corresponding to viable organisms, detected by culturability on agar media and in dilution culture with unamended seawater, was not detectable by DG GE patterns obtained from total cells. Most of the strains isolated by dilution cultures were different from those isolated on solid agar media. These results suggest that studies on the structure of complex marine bacterial communities do not necessarily reflect the physiological heterogeneity of ecologically important populations and may ignore populations present at low relative abundance that can play a key ecological role.

Binder B (2000) Cell cycle regulation and the timing of chromosome replication in a marine Synechococcus (cyanobacteria) during light- and nitrogen-limited growth. Journal of Phycology 36 :120-126

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Cell cycle behavior in the marine Synechococcus strain WH8101 was examined in detail over a wide range of light- and nitrogen-limited growth rates. The presence of bimodal DNA frequency distributions under all conditions confirms that the overlapping rounds of DNA replication that characterize E. coli and other fast-growing prokaryotes are not present in this organism, Although chromosome replication time, C, was constrained to a fairly narrow range of values overall, it nevertheless did vary with growth rate and Limiting factor. Light-limited cells growing at moderate rates had higher C values than did N-limited cells growing at comparable rates (by as much as a factor of 2), As these cells became light saturated, however, C decreased sharply to the level observed under N limitation. The post-replication period, D, decreased monotonically with growth rate under both light and N limitation, approaching a constant value at moderate to high growth rates. Average cell volume at the time of initiation of DNA replication was calculated from the values of C and D, combined with directly measured mean cell volume, and was found to be constant at all growth rates above similar to 0.7 d(-1), This pattern was confirmed by estimates of initiation volume based on flow cytometric light scatter measurements, and suggests that as has been found in other prokaryotic systems, cell mass may play an important role in regulating the timing of chromosome replication in cyanobacteria, Furthermore, because the magnitude of C + D influences average cell mass (given a constant mass at initiation), changes in these parameters (particularly C) may be responsible for the previously reported nonlinear relationship between light-limited growth rate and both RNA cell(-1) and average cell volume.

Blaise C, Gagn F, Bombardier M (2000) Recent developments in microbiotesting and early millennium prospects. Water Air and Soil Pollution 123 :11-23

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Small-scale toxicity testing with microbiotests is a rapidly-expanding component of the field of aquatic toxicology which contributes diverse contamination assessment tools and approaches for a variety of environmental (liquid and solid) media. In this short review on microbiotesting, some of the recent developments conducted under the second St. Lawrence River Action Plan (1993-1998) at the St. Lawrence Centre (Environment Canada, Quebec Region, Montreal) are recalled. These include 1) employing the SOS Chromotest to determine the genotoxic status of major industrial effluents discharging to the St. Lawrence River and their potential impact on downstream biota, 2) developing an algal solid phase assay to predict the toxic potential of freshwater sediments, 3) developing a microplate-based cnidarian assay to screen for toxicity of chemicals and environmental samples, 4) developing an alternative assay to whole fish acute (sub)lethal toxicity testing with the help of rainbow trout primary hepatocytes, 5) developing a microplate-based phagocytosis assay to check for immunocompetence of feral bivalve shellfish and 6) conducting a major investigation to develop a cost-effective multitrophic bioanalytical battery to assess the (geno)toxicity of freshwater sediments. In addition, integrative tools with specific microbiotests were respectively constructed to determine the toxic potential of industrial effluents (PEEP : Potential Ecotoxic Effects Probe) and that of sediments (SED-TOX). Such examples illustrate the diversity of on-going endeavors in the field of small-scale toxicity testing internationally, as further corroborated by recent books entirely dedicated to the subject. It is undeniable that many important challenges still lie ahead for this field early into the third millennium and likely well beyond.

Boelen P, de Boer MK, Kraay GW, Veldhuis MJW, Buma AGJ (2000) UVBR-induced DNA damage in natural marine picoplankton assemblages in the tropical Atlantic Ocean. Marine Ecology-Progress Series 193 :1-9

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UVBR (ultraviolet-B radiation : 280 to 315 nm)-induced DNA damage, measured as cyclobutane pyrimidine dimers (CPDs), was determined in size fractions of natural populations of bacterio- and phytoplankton collected in marine tropical waters. Mean biologically effective UVBR doses in the wind-mixed layer were calculated from DNA dosimeter data. Phytoplankton species composition in these waters was monitored using flow cytometry and pigment analyses. In terms of (divinyl-)chlorophyll a concentrations, prochlorophytes and cyanobacteria comprised the largest fraction of the phytoplankton, except in a eutrophic bay at Curacao an island located in the southern Caribbean. In terms of cell numbers and amount of DNA, small prochlorophytes and marine bacteria dominated. Small but detectable levels of UVBR-induced DNA damage were found at all locations. In general, more DNA damage was found in the small size fraction (0.2 to 1 mu m) than in the larger size fraction (1 to 10 mu m). The greatest amount of damage was found in the small size fraction collected in the central Atlantic Ocean (20 CPDs/10(6) nucleotides), despite the fact that UVBR doses were much higher at other locations. The calculated mean biologically effective UVBR doses in the wind-mixed layer were 2 to 17 times lower as compared with incident UVBR doses. CPD levels determined in cultures of the cyanobacterium Synechococcus sp, subjected to UVBR doses similar to those in the wind-mixed layer corresponded with CPD levels measured in the 1 to 10 mu m fraction in the field. Our results indicate that UVBR vulnerability is size dependent. Furthermore, the low CPD levels observed in these field communities may be explained by the low mean biologically effective doses received by the cells as a result of wind-induced mixing.

Bratvold D, Srienc F, Taub SR (2000) Analysis of the distribution of ingested bacteria in nanoflagellates and estimation of grazing rates with flow cytometry. Aquatic Microbial Ecology 21 :1-12

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The distribution of ingested bacteria in nanoflagellates was assessed to suggest whether or not there are subgroups of grazers with different grazing rates. Several discrete random distributions were compared to the distribution of ingested fluorescently labeled bacteria (FLB) in cultures of Rhynchomonas nasuta and Paraphysomonas vestita. Sample distributions typically fit both the Poisson with extra zeros (Poisson EZ, tested as a truncated Poisson) and negative binomial, but only occasionally fit a Poisson. Both the Poisson EZ and the negative binomial distributions suggest a heterogeneous population composed of subgroups of flagellates with different grazing rates. Although these models provide acceptable mathematical descriptions, their specific biological implications with regard to the number of flagellate subgroups remain to be proven. Based on fit of the distribution of ingested prey to a Poisson EZ, a rapid cytometric method for estimation of grazing rates on FLB is presented. The method uses changes in the probability of grazers not ingesting FLB during short incubations (ca 15 min) to estimate the Poisson parameter and the fraction of extra zeros, from which the average grazing rate is calculated. Grazing rates determined by microscopy and by this cytometry method were similar. Frequency distributions of cytometric histograms of fluorescent microspheres in grazers suggest that both the Poisson EZ and negative binomial models are simplifications of a more complex distribution of grazing rates.

Brookes JD, Geary SM, Ganf GG, Burch MD (2000) Use of FDA and flow cytometry to assess metabolic activity as an indicator of nutrient status in phytoplankton. Marine and Freshwater Research 51 :817-823

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This study reports the use of a technique to determine nutrient limitation of cultured and natural phytoplankton. The technique, an FDA-activity assay, which is usually used to assess cell viability, was used to measure metabolic activity in response to nutrient addition ; the metabolic activity of phytoplankton was determined as the rate of hydrolysis of fluorescein diacetate (FDA), by intracellular esterases, to fluorescein, which was detected using a flow cytometer. Replacement of the limiting nutrient to nitrogen- or phosphorus-limited cultures and field populations resulted in an increase in metabolic activity that was detectable 24 h after nutrient addition. By flow cytometry, the natural phytoplankton community can be divided into different taxonomic groups ; the response of these to FDA could be determined individually to allow identification of the nutrients limiting each type of phytoplankton. This would be more specific than the assessment of a whole-community response, which may mask subtle differences among taxa.

Brousseau P, Pellerin J, Morin Y, Cyr D, Blakley B, Boermans H, Fournier M (2000) Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals. Toxicology 142 :145-156

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The effectiveness of toxicology biomonitoring programs could be improved by the addition of sensitive biomarkers. In this study the cell viability and sensitivity of phagocytic function of phagocytes from bivalves (Mya arenaria) to selected heavy metals were measured by flow cytometry, a novel approach. Hemocytes (phagocytes) collected from bivalves by puncture of the posterior adductor muscle were incubated in vitro for 18 h in hemolymph containing 10(-9)-10(-3) M of cadmium chloride, zinc chloride, mercuric chloride, methylmercury chloride or silver nitrate, before determining their capacity to phagocytose fluorescent latex beads by ;flow cytometry. Heterogeneity of the hemocyte cell population was determined by forward scatter (FSC) and side scatter (SSC) cytometric profile which showed two distinct cell populations. At low doses (10(-9), 10(-8) M), all the metal compounds studied stimulated phagocytic activity except silver nitrate. At higher levels of exposure (10(-6), 10(7) M), all metals caused a significant concentration-related decrease in hemocyte phagocytosis activity. From the concentration of each metal inducing 50% suppression (IC50) of the phagocytic activity, the immunotoxic potential of metals with respect to phagocytic function can be ranked in the following increasing order : ZnCl2 < CdCl2 < AgNO3 < HgCl2 < CH3HgCl. Parallel analysis of hemocyte viability showed that suppression of phagocytosis by heavy metals was not solely related to a. decreased cell viability. These results reveal the high but different degree of sensitivity of the phagocytosis activity of bivalves with respect to heavy metals, as measured by flow cytometry, and demonstrate that flow cytometry is a potentially useful tool in ecotoxicological monitoring. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Buma AGJ, van Oijen T, van de Poll W, Veldhuis MJW, Gieskes WWC (2000) The sensitivity of Emiliania huxleyi (Prymnesiophycea) to ultraviolet-B radiation. Journal of Phycology 36 :296-303

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Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR : 280-315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR : at a daily weighted UVBR dose of only 400 J.m(-2).d(-1) (BEDDNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pig pigments had increased. The UVBR vulnerability of huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA : the CG content of this E huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity, It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.

Button DK, Robertson B (2000) Effect of nutrient kinetics and cytoarchitecture on bacterioplankter size. Limnology and Oceanography 45 :499-505

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Expanded specific affinity theory specifies the advantages of small dry mass and dilute cytoarchitecture in impoverished systems. For marine samples, bacterioplankton mean dry mass, according to flow cytometry, was near 24.5 fg cell(-1). Conversion to volume with buoyant density gave a cell volume of 0.124 mu m(3). Total DNA was 2.9 fg cell(-1). This compared with the size of a single genome of a small extinction-culture isolate, Sphingomonas sp. RB2256, of 3.96 fg or 3.6 Mb. The genome size of such isolates and other cultures decreased with metabolic simplicity. It was found that bacterioplankton could be exposed to radiolabeled amino acids and then sorted for size and that the specific affinities of the fraction of small organisms were as great as the fraction of large cells. Size, DNA, and metabolic-complexity distributions were concordant with the concepts that cell volume approaches a minimum set by sufficient space for the smallest genome that can provide sufficient information for competitive dissolved-nutrient acquisition, and that space requirements are further alleviated by the expression of few cytoplasmic-enzyme molecules in each of the various pathways and a dilute cytoplasm. Bacterioplankton approached a minimum genome size of 1.7 Mb with a minimum cell volume of about 0.06 mu m(3) and a DNA content of 16% dry weight. The property of small dry mass with a low DNA content was common in in situ bacteria but absent from cultivated representatives, which led to speculation that failure to grow in the laboratory is related to missing regulatory information.

Casotti R, Brunet C, Aronne B, d’Alcala MR (2000) Mesoscale features of phytoplankton and planktonic bacteria in a coastal area as induced by external water masses. Marine Ecology-Progress Series 195 :15-27

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The effects of different water masses and their physical structures on phytoplankton and bacteria were investigated in a coastal area of the Mediterranean Sea, the Gulf of Naples (Italy) on 9 and 10 November 1995. A small eddy (8 km in diameter), generated from offshore waters, occupied the inner part of the Gulf at surface, while the Modified Atlantic Water (MAW) intruded between 60 and 80 m depth. Phytoplankton communities as identified by HPLC pigment analysis (total) and flow cytometry (ultraphytoplankton) showed peculiarities in the different subsystems. Cryptophytes dominated by pigment biomass were everywhere, while diatoms were more abundant at the coast and nanoflagellates at offshore stations. In the MAW, a peculiar phytoplankton community, different to that in the other water masses, occurred, and 2 populations of prochlorophytes were distinguished based on scatter and red fluorescence observations. Total bacterial numbers were significantly correlated to chlorophyll a (chl a) concentrations. Different populations of heterotrophic bacteria were identified based on apparent DNA content. Percentages of 2 such populations were significantly correlated to total chi a. Therefore, phytoplankton appears to regulate not only bacterial quantity but also composition. Although our study area was characterized by a marked oligotrophy, heterotrophic bacterial biomass never exceeded autotrophic biomass, as derived using constant conversion factors of bacteria and chi a to C. Therefore, no inverted pyramid was observed and bacterial biomass was still controlled by phytoplankton biomass. Mesoscale variability is of great relevance for energy and matter budgets of coastal areas and its study is therefore fundamental. For example, eddies can be very important in coastal areas, influencing distribution of nutrients and organisms. Our approach, coupling HPLC and flow cytometry, represents a valuable tool for obtaining information on both phytoplankton composition and ecophysiology on a large number of samples, necessary for a valid representation of the space scales involved.

Chen F, Binder B, Hodson RE (2000) Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR. FEMS Microbiol Lett 184 :291-296


Prokaryotic in situ RT-PCR was coupled with flow cytometry to detect mRNA transcripts of the toluene dioxygenase (todC1) gene in intact cells of the bacterium Pseudomonas putida F1. Recovery efficiency of fixed cells over the course of the entire in situ detection procedure was approximately 81% for both P. putida F1 and AC10R cells. It appeared that lysozyme treatment and PCR thermal cycling were the steps responsible for most of observed cell loss. Bacterial cells expressing the todC1 gene could be discriminated from negative control cells of the same size based on flow cytometrically-measured fluorescence and forward angle light scatter. According to flow cytometric analysis, the fluorescence intensity of positive cells was 4-5 times brighter than that of negative cells. The combination of flow cytometry and a prokaryotic in situ reverse transcription-PCR (RT-PCR) approach make possible the rapid detection and enumeration of functional (based on mRNA) populations of microbial cells.

Collier JL (2000) Flow cytometry and the single cell in phycology. Journal of Phycology 36 :628-644

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Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell-level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in-flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of non-planktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single-cell taxonomic and physiological in-formation to be garnered for a variety of algae, both in culture and in nature.

Corzo A, Morillo JA, Rodriguez S (2000) Production of transparent exopolymer particles (TEP) in cultures of Chaetoceros calcitrans under nitrogen limitation. Aquatic Microbial Ecology 23 :63-72

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Transparent exopolymer particles (TEP) are considered to be generated abiotically from dissolved extracellular polysaccharides released mainly by phytoplankton. TEP may affect the aggregation rate of particles and therefore influence the flux of organic carbon to the deep ocean. The role of NO3- limitation in the production of TEP by the marine diatom Chaetoceros calcitrans was investigated. C. calcitrans was grown in batch cultures with different initial nitrate concentrations (25, 75, 150, 250 and 450 mu mol l(-1)). Nitrate affected the production of TEP in 2 distinct ways. The initial specific growth rate and maximum concentration of biomass las estimated by chlorophyll a, cell number, total particulate carbon or total particulate nitrogen) reached in every culture was directly dependent on the initial NO3- concentration. Maximum TEP concentration followed this trend, and was significantly linearly correlated with several biomass-related variables and with the initial NO3- concentration. However, despite the general trend of direct covariation between TEP concentration and phytoplankton biomass, NO3- has a more specific effect. A close examination of the exponential phase shows that the net production of TEP per biomass was higher in N-limited cultures. In the N-sufficient cultures, during Day 2, there was even a decrease in the concentration of TEP per unit of biomass with respect to the inoculum. Our results support the hypothesis that, under N-limitation, a large proportion of the photosynthetically fixed carbon is channeled to TEP. The concentration of carbohydrate in the particulate fraction (PCH) was larger in N-limited cultures. The effect was clearer during exponential growth. In this phase there was a good agreement between the response of TEP and PCH to N limitation. Unlike TEP, PCH decreased sharply during the stationary and senescent phases. The decrease of PCH was associated with an increase in dissolved organic carbon and an increase in the number of bacteria in the cultures.

Denis M, Martin V, Andersen V (2000) Short-term variations of the vertical distribution of cyanobacteria in the open Mediterranean Sea. Scientia Marina 64 :157-163

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A general study of biogeochemical processes (DYNAPROC cruise, May 1995) was conducted at a station in the open northwestern Mediterranean Sea where horizontal advection was weak. During this experiment, short-term variations of the vertical distribution of cyanobacteria were investigated and a possible link with diel vertical migration was considered. Consistently, the experimental work was conducted at a time scale of a few hours. Cyanobacteria were the most abundant population in the pico- and nanoplankton size-class. Their vertical distribution was monitored over 36 h with a frequency of 4 h. With such a time resolution, they experienced a single grazing event during daytime and, at night, they were heavily grazed when the upper layers were occupied by organisms migrating from the aphotic zone. The corresponding integrated (0-90 m) losses of cyanobacteria during the night amounted to 534 mg C m(-2). Though daytime grazing is most likely due to nanoflagellates and microzooplankton, night-time grazing could significantly involve migrant macrozooplankton organisms and sustain a periodic export of organic matter. This study stresses the importance of the potential export of organic matter as a consequence of the diel vertical migration, export that could not be accounted for by measurements at longer time scale.

Domingo JWS, Harmon S, Bennett J (2000) Survival of Salmonella species in river water. Current Microbiology 40 :409-417

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The survival of four Salmonella strains in river water microcosms was monitored by culturing techniques, direct counts, whale-cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytometry. Plate counts of bacteria resuspended in filtered and untreated river water decreased several orders of magnitude within the first week of incubation, while they did not decrease as rapidly in autoclaved water. In situ hybridization studies suggested a rapid decrease in ribosomal content, as determined by the drastic decrease in the number of detectable cells after 72 h. In contrast, direct counts remained relatively constant during 45 days in all microcosoms. Although the culturable counts of two bacterial strains in filtered water after 31 days represented approximately 0.001% of the total counts, direct viable counts and resuscitation studies with a dilution series suggested that the number of viable bacteria was at least four orders of magnitude higher. Additionally, notable changes in forward scatter and in nucleic acid content were observed only after 4 h of nutrient amendments by flow cytometry. However, cells from the resuscitation experiments did not grow on solid media unless cell-free supernatant from viable cultures was added during the resuscitation period. The results in this study suggest the presence of a not immediately culturable status in Salmonella.

Dubelaar GBJ, Gerritzen PL (2000) CytoBuoy : a step forward towards using flow cytometry in operational oceanography. Scientia Marina 64 :255-265

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While the performance of biological sensors in real time monitoring networks is limited to bulk Values like chlorophyll fluorescence, in practice the implementation of automated phytoplankton taxonomy remains a remote option. Aiming to reduce this gap we developed a flow cytometer called CytoBuoy for autonomous in situ operation, for instance in a moored buoy with wireless data transfer. Although not comparable to microscopy, flow cytometers detect and count particles allowing a limited level of particle characterization based on the light scatter and fluorescence properties of the individual particles. CytoBuoy analyses a large size range of particles, typical for marine coastal zones and fresh waters. The ’field’ design implies a tradeoff between the accuracy and versatility of laboratory flow cytometers and the qualities needed for trouble free autonomous operation in situ. The optics and electronics however were designed for maximal reflection of the particle morphology in the measured signals. Whereas standard cytometers reduce these to single peak or area ’listmode’ numbers, the signal courses are preserved fully by CytoBuoy and transferred to the computer as raw data, which allows more extended morphological analysis. Extended field tests will have to show how the system holds in various environments and weather conditions.

Dubelaar GBJ, Jonker RR (2000) Flow cytometry as a tool for the study of phytoplankton. Scientia Marina 64 :135-156

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An overview is presented on flow cytometry as a tool for counting, analysis and identification of phytoplankton species and groups. The paper covers basics on the analysis technique and instrumentation such as the measuring principle, the type of instrument, limitations and pitfalls with phytoplankton samples and sample handling and preprocessing. Possibilities of the measured entities are discussed, roughly divided in light scatter and related parameters, the endogenous fluorescence and exogenous fluorescence, followed by a discussion on the actual applications such as phytoplankton abundance analysis, ecology and physiology research and monitoring of particle size and biomass. In addition to a limited literature review, we tried to assess how flow cytometry is used in routine laboratory practice and monitoring operations. Therefore, a questionnaire was sent out via email to 47 scientists at 43 institutes known to us as involved in flow cytometric analysis of phytoplankton. In total, 19 scientists responded. Specific survey results are included in italic print whereas some more general answers were integrated in the overview.

Dusenberry JA (2000) Steady-state single cell model simulations of photoacclimation in a vertically mixed layer : implications for biological tracer studies and primary productivity. Journal of Marine Systems 24 :201-220

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A numerical single-cell photoacclimation-diffusion model was constructed and used to develop criteria regarding the use of individual phytoplankton cells as tracers for vertical mixing and to illustrate how rates of vertical mixing might affect phytoplankton physiology. Both first-order and logistic representations of photoacclimation kinetics were used. Steady state was assumed for simplicity and to provide a starting point for further investigations. The modeled variance and higher moments (within a phytoplankton population) of a generic photoacclimative parameter all show trends, which are diagnostic of mixing rates and/or boundary effects. This allowed the establishment of criteria by which frequency distributions of phytoplankton physiological properties (e.g., cell fluorescence) might be used as indicators of vertical mixing. The same model can be used to predict the effects of vertical mixing on phytoplankton productivity and growth. Application of the model to both photosynthesis and carbon to chlorophyll ratios suggested that a combination of vertical mixing and hysteresis las represented in the logistic model of photoacclimation) in acclimation kinetics can enhance specific growth rates of phytoplankton. This enhanced growth occurred as a result of mixing-induced variation in carbon to chlorophyll ratios and is in contrast to chlorophyll-specific productivity, which was maximal at low mixing rates. Differential rates of photoacclimation to upward vs, downward shifts in irradiance, may enable phytoplankton cells to better survive in a turbulent environment. (C) 2000 Elsevier Science B.V. All rights reserved.

Dusenberry JA, Olson RJ, Chisholm SW (2000) Field observations of oceanic mixed layer dynamics and picophytoplankton photoacclimation. Journal of Marine Systems 24 :221-232

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To explore the relationship between mixing dynamics in the surface ocean and picophytoplankton optical properties, we measured the distributions of single cell fluorescence in Prochlorococcus populations throughout a diel cycle. Two time series were conducted in the N. Atlantic, and both showed a shoaling of the mixed layer, due to surface warming or a rain-formed surface layer. These dynamics, coupled with the diel cycle of solar irradiance, drove the development of a depth gradient in mean red fluorescence of Prochlorococcus, due to photoacclimation, in the newly-stratified layer. Furthermore, the frequency distribution of single-cell fluorescence within field populations appears to have responded to changing mixing and photoacclimation dynamics, with photoacclimation in the absence of strong mixing generally resulting in a reduced variance in fluorescence within sample populations. Nighttime mixing in the absence of photoacclimation reversed this process and resulted in increased variation of single-cell fluorescence. Departures from normality in observed distributions suggest vertical mixing time-scales that are slightly longer than time-scales of photoacclimation. The behavior of the mean and variance, and possibly the third and fourth moments, of single-cell optical properties is consistent with photoacclimation in response to the physical dynamics. (C) 2000 Elsevier Science B.V. All rights reserved.

Eilers H, Pernthaler J, Amann R (2000) Succession of pelagic marine bacteria during enrichment : a close look at cultivation-induced shifts. Appl Environ Microbiol 66 :4634-4640


Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S-) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S-, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant gamma-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable gamma-proteobacteria could thus be described as a "feast and famine" existence involving different activation levels of substrate concentration.

Endo H, Nakayama J, Hayashi T (2000) Application of flow cytometry to environmental control in marine aquaculture. Materials Science & Engineering C-Biomimetic and Supramolecular Systems 12 :83-88

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The application of flow cytometry (FCM) technique for rapid detection of pathogenic bacteria for fish in marine aquaculture was described. To determine the number of viable bacteria cells, a fluorescein diacetate (FDA) was used. The difference in fluorescence scattergram between viable and dead cells was observed. The FCM method provided rapid determination of cell number of viable bacteria. A good correlation was observed between the values determined by the FCM method and the colony counting method in the range of 10(5)-10(8) cells/ml, One FCM assay could be completed within 1 min. The FCM technique was also applied to rapid detection of pathogenic bacteria (Lactococcus garvieae). An antiserum against L. garvieae was prepared and its immunological property was examined. The detection of L. garvieae in cell suspensions contaminated with Escherichia coli was carried out. One FCM assay could be completed within 2 min and the total assay time including the preparation of bacterial sample was within 3 h. (C) 2000 Elsevier Science S.A. All rights reserved.

Franqueira D, Orosa M, Torres E, Herrero C, Cid A (2000) Potential use of flow cytometry in toxicity studies with microalgae. Sci Total Environ 247 :119-126


Cytotoxic effects of aquatic pollutants on microalgae are very heterogeneous, and they are influenced by environmental conditions and the test species. Stress produced by copper or paraquat addition to the culture medium of two microalgae was analysed by flow cytometry. Parameters assayed were : cell volume, chlorophyll a fluorescence and cell viability. The variety of results obtained in the present study reveals that flow cytometry is a useful tool in the toxicity tests with microalgae, both marine and freshwater species, and for different kind of pollutants.

Fuchs BM, Glockner FO, Wulf J, Amann R (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66 :3603-3607


Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.

Fuchs BM, Zubkov MV, Sahm K, Burkill PH, Amann R (2000) Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques. Environ Microbiol 2 :191-201


Dilution cultures are a common technique for measuring the growth of bacterioplankton communities. In this study, the taxonomic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK). Bacterial abundances as well as protein and DNA content were closely monitored by flow cytometry. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cells sorted from the dilution cultures based on their protein and DNA content. As expected, a rapid activation of bacteria occurred. However, molecular techniques showed that the community developed in the dilution culture within 1 day was significantly different from that in the original water samples. Whereas in the original samples, cells detectable by FISH were dominated by members of the Cytophagal Flavobacterium (CF) cluster, in dilution cultures, gamma-proteobacteria accounted for the majority of cells detected, followed by alpha-proteobacteria. An actively growing and an apparently non-growing population with average cellular protein contents of 24 and 4.5 fg respectively, were sorted by flow cytometry. FISH indicated mostly gamma- (64%) and alpha-proteobacteria (33%) in the first active fraction and 78% members of the CF cluster in the second fraction. Sequencing of DGGE bands confirmed the FISH assignments of the latter two groups. The data presented clearly show that even relatively short-term dilution experiments do not measure in situ growth, but rather growth patterns of an enrichment. Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid method of analysing the taxonomic composition of marine bacterioplankton.

Garrison DL, Gowing MM, Hughes MP, Campbell L, Caron DA, Dennett MR, Shalapyonok A, Olson RJ, Landry MR, Brown SL, Liu HB, Azam F, Steward GF, Ducklow HW, Smith DC (2000) Microbial food web structure in the Arabian Sea : a US JGOFS study. Deep-Sea Research Part Ii-Topical Studies in Oceanography 47 :1387-1422

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One of the main objectives of the Joint Global Ocean Flux Studies (JGOFS) program is to develop an understanding of the factors controlling organic carbon production in the ocean and the time-varying vertical flux of carbon from surface waters (US JGOFS (1990) US JGOFS Planning Report Number 11 ; Sarmiento and Armstrong (1997) US JGOFS Synthesis and Modeling Project Implementation Plan). A considerable amount of evidence suggests that carbon cycling and the potential for exporting carbon from ocean systems is a function of food web structure. As part of the US JGOFS Arabian Sea Studies, the biomass of planktonic organisms, ranging from heterotrophic bacteria through microplankton-sized organisms, was estimated using a variety of methods including flow cytometry and microscopy. This is a first attempt to combine biomass data from a number of sources, evaluate the structure of the food web, examine changes in food web structure in relation to seasonal or spatial features of the study area, and look for indications of how changing structure affects carbon-cycling processes. Biomass in the upper 100 m of the water column ranged from approximately 1.5 to > 5.2 gC m(-2). Heterotrophic bacteria (Hbac) made up from 16 and 44% of the biomass ; autotrophs comprised 43-64% ; and the remainder was made up of nano- and microheterotrophs. Autotrophs and nano- and microheterotrophs showed a general pattern of higher values at coastal stations, with the lowest values offshore. Heterotrophic bacteria (Hbac) showed no significant spatial variations. The Spring Intermonsoon and early NE Monsoon were dominated by autotrophic picoplankton, Prochlorococcus and Synechococcus. The late NE Monsoon and late SW Monsoon periods showed an increase in the larger size fractions of the primary producers. At several stations during the SW Monsoon, autotrophic microplankton, primarily diatoms and Phaeocystis colonies, predominated. Increases in the size of autotrophs were also reflected in increasing sizes of nano- and microheterotrophs. The biomass estimates based on cytometry and microscopy are consistent with measurement of pigments, POC and PON. Changes in community structure were assessed using the percent similarity index (PSI) in conjunction with multidimensional scaling (MDS) or single-linkage clustering analysis to show how assemblages differed among cruises and stations. Station clustering reflected environmental heterogeneity, and many of the conspicuous changes could be associated with changes in temperature, salinity and nutrient concentrations. Despite inherent problems in combining data from a variety of sources, the present community biomass estimates were well constrained by bulk measurements such as Chi a, POC and PON, and by comparisons with other quantitative and qualitative studies. The most striking correlation between Food web structure and carbon cycling was the dominance of large phytoplankton, primarily diatoms, and the seasonal maxima of mass flux during the SW Monsoon. High nutrient conditions associated with upwelling during the SW Monsoon would explain the predominance of diatoms during this season. The sinking of large, ungrazed diatom cells is one possible explanation for the flux observations, but may not be consistent with the observation of concurrent increases in larger microzooplankton consumers (heterotrophic dinoflagellates and ciliates) and mesozooplankton during this season. Food-web structure during the early NE Monsoon and Intermonsoons suggests carbon cycling by the microbial community predominated. (C) 2000 Elsevier Science Ltd. All rights reserved.

Gasol JM, Del Giorgio PA (2000) Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Scientia Marina 64 :197-224

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Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as Fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

Gilbert M, Domin A, Becker A, Wilhelm C (2000) Estimation of primary productivity by chlorophyll a in vivo fluorescence in freshwater phytoplankton. Photosynthetica 38 :111-126

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Primary productivity in marine waters is widely estimated by the measurements of C-14 incorporation, the underwater light climate, and the absorption spectra of phytoplankton. In bio-optical models the quantum efficiency of carbon fixation derived from C-14 incorporation rates, the photosynthetically absorbed radiation derived from the underwater light climate, and the phytoplankton absorption spectra are used to calculate time- and depth-integrated primary productivity. Due to the increased sensitivity of commercially available fluorometers, chlorophyll a in vivo fluorescence became a new tool to assess the photosynthetic activity of phytoplankton. Since fluorescence data yield only relative photosynthetic electron transport rates, a direct conversion into absolute carbon fixation rates is not possible. Here, we report a procedure how this problem can be adressed in freshwater phytoplankton. We adapted a marine bio-optical model to the freshwater situation and tested if this model yields realistic results when applied to a hypertrophic freshwater reservoir. Comparison of primary productivity derived from C-14 incorporation to primary productivity derived from Chi a fluorescence showed that the conversion of fluorescence data into carbon fixation rates is still an unsolved problem. Absolute electron transport rates calculated from fluorescence data tend to overestimate primary production. We propose that the observed differences are caused mainly by neglecting the package effect of pigments in phytoplankton cells and by non-carbon related electron flow (e.g., nitrogen fixation). On the other hand, the C-14 incorporation rates can be artificially influenced by ’’bottle effects", especially near the water surface, where photoinhibition, photorespiration, and Mehler reaction can play a major role.

Goericke R, Olson RJ, Shalapyonok A (2000) A novel niche for Prochlorococcus sp in low-light suboxic environments in the Arabian Sea and the Eastern Tropical North Pacific. Deep-Sea Research Part I-Oceanographic Research Papers 47 :1183-1205

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Prochlorococcus marinus is present in all tropical and subtropical oceans, where it is often found throughout the euphotic zone, contributing significantly to phytoplankton biomass and primary production and growing at rates comparable to those of other picoplankters. Clearly, Prochlorococcus and eucaryotic picoplankton share significant niche dimensions in the open ocean. Here we report the discovery of populations of Prochlorococcus in layers below the oxyclines of the oxygen minimum zones of the Arabian Sea and the Eastern Tropical North Pacific off Mexico. The unusual aspects of these populations are that these were at times virtual monoalgal cultures found at a depth of 80 to 140 m, often below the euphotic zone, where irradiance ranged from less than 0.1 to 2% of the surface irradiance (I-o), The pigment complement of these deep populations was characterized in detail, The previously unidentified Chl-c-like pigment of Prochlorococcus is Mg-3,8-divinylpheoporphyrin a(5) monomethylester, The carotenoid complement of populations in these deep layers was similar to that of cultured Prochlorococcus strains, except for high concentrations of a 7’,8’-dihydro-derivative of zeaxanthin, quite likely parasiloxanthin, Even though cellular concentrations of pigments were very high in these populations, suggesting acclimation to low irradiance, ambient Light experienced by these populations in the Arabian Sea, <0.1% I-o, may not have been sufficient to support normal photoautotrophic growth. Off Mexico these deep Prochlorococcus populations were located at 0.2 and 2% I-o isolumes, an irradiance likely sufficient for slow growth. Environmental conditions in these layers, except for concentrations of oxygen, are similar to those found at and below the subsurface chlorophyll maxima of the subtropical central gyres. As only Prochlorococcus thrives in these layers but Prochlorococcus and eucaryotic picoplankton coexist in and below subsurface chlorophyll maxima. we conclude that the low oxygen concentrations at the deep Prochlorococcus maxima are the determining factor, but we are not able to identify any specific physiological functions that are affected by low oxygen concentrations in eucaryotes but not Prochlorococcus. (C) 2000 Elsevier Science Ltd. All rights reserved.

Goodwin TJ, Coate-Li L, Linnehan RM, Hammond TG (2000) Selected contribution : a three-dimensional model for assessment of in vitro toxicity in balaena mysticetus renal tissue. J Appl Physiol 89 :2508-2517


This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth’s surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.

Jochem FJ (2000) Probing the physiological state of phytoplankton at the single-cell level. Scientia Marina 64 :183-195

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Probing the physiological state of phytoplankton at the single-cell level provides valuable insight in ecological studies as well as in environmental monitoring of pollution or UV impacts. This paper reviews the recent progress in assessing the physiological state of phytoplankton with flow cytometry by inherent cell properties such as cell size and chlorophyll autofluorescence, specific fluorescent dyes, and newly developed molecular probes and enzyme substrates. It is reported how nitrogen and iron limitation as well as the effect of copper pollution could be derived from changes in cell inherent properties. Effects of Cu were also recorded by monitoring cell membrane potentials and esterase activity. Photosynthetic capacity of algae was assessed by changes in chlorophyll fluorescence with the electron transport inhibitor DCMU, by a cytometric adaptation of the pump-and-probe approach, and molecular probes for Rubisco. Antibodies were also applied to mark non-terminal stages in the cell DNA replication cycle, to detect non-proliferating cells, to assess DNA damage caused by UV-B radiation and to quantify diatom stickiness. Fluorescein diacetate proved useful to discriminate metabolically active from inactive cells and to reveal strategies of dark survival in algae. The activity of alkaline phosphatase was recorded by a new fluorigenic substrate ELF, and polyclonal antibodies against nitrate reductase (NR) provided measurements of the NR abundance. An outlook will show how recent developments in molecular probes might affect the future analysis of marine ecosystems and their communities.

Jochem FJ, Smith GJ, Gao Y, Zimmerman RC, Cabello-Pasini A, Kohrs DG, Alberte RS (2000) Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum. Cytometry 39 :173-178


BACKGROUND : The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS : Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS : NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS : The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.

Jonker R, Groben R, Tarran G, Medlin L, Wilkins M, Garcia L, Zabala L, Boddy L (2000) Automated identification and characterisation of microbial populations using flow cytometry : the AIMS project. Scientia Marina 64 :225-234

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The AIMS (Automatic Identification and characterisation of Microbial populationS) project is developing and integrating flow cytometric technology for the identification of microbial cell populations and the determination of their cellular characteristics. This involves applying neural network approaches and molecular probes to the identification of cell populations, and deriving and verifying algorithms for assessing the chemical, optical and morphometric characteristics of these populations. The products of AIMS will be calibrated data, protocols, algorithms and software designed to turn flow cytometric observations into a data matrix of the abundance and cellular characteristics of identifiable populations. This paper describes the general approach of the AIMS project, with details on the application of artificial neural nets and rRNA oligonucleotide probes.

Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes and Infection 2 :1523-1535

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A wide diversity of fluorescent probes is currently available to assess the physiological state of microorganisms. The recent development of techniques such as solid-phase cytometry, the increasing sensitivity of fluorescence tools and multiparametric approaches combining taxonomic and physiological probes have improved the effectiveness of direct methods in environmental and industrial microbiology. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Jugnia LB, Richardot M, Debroas D, Sime-Ngando T, Devaux J (2000) Variations in the number of active bacteria in the euphotic zone of a recently nodded reservoir. Aquatic Microbial Ecology 22 :251-259

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Using the CTC (5-cyano-2,3-ditolyl tetrazolium chloride) method, we estimated the active fraction of bacterioplankton in the euphotic zone of the recently flooded Sep Reservoir, France, with the aim of evaluating its importance and dynamics in relation to temperature and some components of dissolved organic matter (DOM) (i.e. dissolved combined amino acids : DCAA ; dissolved free monosaccharides : DFCHO ; dissolved combined monosaccharides : DCCHO), and its metabolic significance in conjunction with a bacterial growth indicator : C-14-glucose uptake. From our results, it appears that only a small fraction (0.04 to 3.23 %) of total bacterial count in this reservoir was metabolically active. Recorded correlations and multivariate regression analysis suggest that temporal variations in the number of active bacteria are mainly (80 %) governed by temperature, together with the high concentrations of those components of DOM, which were present in this ecosystem but which apparently were not always available for bacterial metabolism. We also found a positive relation between the number of active bacteria and the potential heterotrophic activity, which suggest that CTC-positive bacteria are responsible for the bulk of bacterial community metabolism in this recently formed reservoir.

Kachel V, Wietzorrek J (2000) Flow cytometry and integrated imaging. Scientia Marina 64 :247-254

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It is a serious problem to relate the results of a flow cytometric analysis of a marine sample to different species. Images of particles selectively triggered by the flow cytometric analysis and picked out from the flowing stream give a valuable additional information on the analyzed organisms. The technical principles and problems of triggered imaging in flow are discussed, as well as the positioning of the particles in the plane of focus, freezing the motion of the quickly moving objects and what kinds of light sources are suitable for pulsed illumination. The images have to be stored either by film or electronically. The features of camera targets and the memory requirements for storing the image data and the conditions for the triggering device are shown. A brief explanation of the features of three realized flow cytometric imaging (FCI) systems is given : the Macro Flow Planktometer built within the EUROMAR MAROPT project, the Imaging Module of the European Plankton Analysis System, supported by the MAST II EurOPA project and the most recently developed FLUVO VI universal flow cytometer including HBO 100- and laser excitation for fluorescence and scatter, Coulter sizing as well as bright field and and phase contrast FCI.

Lopez-Amoros R, Comas J, Garcia MT, Vives-Rego J (2000) Flow cytometric analysis of a marine LAS-degrading consortia. Microbios 101 :23-36


The specific nucleic acid fluorochrome SYTO-13 was used in flow cytometric analysis to assess changes in the density and heterogeneity of marine bacterial populations which biodegrade linear alkylbenzene sulphonate (LAS). Seawater samples with LAS and incubated in the laboratory (20 degrees C, 100 rpm, 30 days) were used to monitor LAS-degrading consortia. Flow cytometric studies and culture methods were used to characterize the LAS degrading bacterioplankton consortia. Fluorescence and scatter signals enabled us to define three regions (R1, R2 and R3) in the dual parameter cytograms. The distribution of the bacterial counts in these regions allowed us to monitor the formation and evolution of the consortia.

Loret P, Pastoureaud A, Bacher C, Delesalle B (2000) Phytoplankton composition and selective feeding of the pearl oyster Pinctada margaritifera in the Takapoto lagoon (Tuamotu Archipelago, French Polynesia) : in situ study using optical microscopy and HPLC pigment analysis. Marine Ecology-Progress Series 199 :55-67

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The in situ diet of the pearl oyster Pinctada margarifera was determined in the lagoon of Takapoto Atoll by comparing the phytoplankton composition of water and bivalve gut contents using 2 different methods, optical microscopy and HPLC pigment analysis. In order to evaluate the available food resources for pearl oysters in the water column, a new method for estimating the pigment/chlorophyll a (chl a) ratio (based on an inverse analysis) was developed which allowed us to determine the contribution of the main phytoplanktonic groups in terms of chi a. In the water, picocyanobacteria and nanoflagellates predominated, the latter being mainly chlorophytes and prymnesiophytes. Comparisons between the results obtained by the 2 methods of investigation indicated that most of the dinoflagellates are unpigmented and, therefore, heterotrophic. An examination of the gut contents showed that picocyanobacteria were only weakly ingested by the oyster and, thus, nanoflagellates constituted the main food resource. Cryptophytes, although poorly represented in the water, were preferentially ingested. Chlorophytes were inefficiently digested since they were found alive and motile in the faeces of the oyster. The ecological implications of this feeding behaviour are discussed.

Manire CA, Rhinehart HL (2000) Use of human recombinant erythropoietin for the treatment of nonregenerative anemia in a rough-toothed dolphin (Steno bredanensis). J Zoo Wildl Med 31 :157-163


Erythropoietin, a glycoprotein growth hormone that is produced primarily in the kidneys, promotes mitosis and survival of erythroid progenitors. The recent synthesis of the human form of the hormone by recombinant technology has provided a new therapeutic option, which is being used in both human and veterinary medicine for treatment of various anemias. A mature male rough-toothed dolphin, Steno bredanensis, was treated with human recombinant erythropoietin in an attempt to resolve a nonregenerative anemia. Two i.m. injections 48 hr apart were associated with an almost immediate increase in circulating immature reticulocytes, total reticulocytes, and nucleated erythrocytes. Over the next several weeks, the hematocrit, hemoglobin, and erythrocyte counts returned to normal, and the animal was subsequently released back into the wild. Endogenous erythropoietin concentrations were determined for this animal as well as three other conspecifics by an enzyme-linked immunosorbent assay for human erythropoietin. These measurements showed circulating erythropoietin concentrations (5-20+ mU/ml) similar to those of most other mammals. This study suggests that human recombinant erythropoietin can be safely and effectively used in this species and may have applicability to other cetacean species for the treatment of nonregenerative anemia. Caution should be exercised during long-term use because production of antibodies to human recombinant and endogenous erythropoietin may lead to potentially serious side effects.

Meyers SP (2000) Developments in aquatic microbiology. Int Microbiol 3 :203-211


Major discoveries in marine microbiology over the past 4-5 decades have resulted in the recognition of bacteria as a major biomass component of marine food webs. Such discoveries include chemosynthetic activities in deep-ocean ecosystems, survival processes in oligotrophic waters, and the role of microorganisms in food webs coupled with symbiotic relationships and energy flow. Many discoveries can be attributed to innovative methodologies, including radioisotopes, immunofluorescent-epifluorescent analysis, and flow cytometry. The latter has shown the key role of marine viruses in marine system energetics. Studies of the components of the "microbial loop" have shown the significance of various phagotrophic processes involved in grazing by microinvertebrates. Microbial activities and dissolved organic carbon are closely coupled with the dynamics of fluctuating water masses. New biotechnological approaches and the use of molecular biology techniques still provide new and relevant information on the role of microorganisms in oceanic and estuarine environments. International interdisciplinary studies have explored ecological aspects of marine microorganisms and their significance in biocomplexity. Studies on the origins of both life and ecosystems now focus on microbiological processes in the marine environment. This paper describes earlier and recent discoveries in marine (aquatic) microbiology and the trends for future work, emphasizing improvements in methodology as major catalysts for the progress of this broadly-based field.

Moon-van der Staay SY, van der Staay GWM, Guillou L, Vaulot D, Claustre H, Medlin LK (2000) Abundance and diversity of prymnesiophytes in the picoplankton community from the equatorial Pacific Ocean inferred from 18S rDNA sequences. Limnology and Oceanography 45 :98-109

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Picoplankton, i.e., cells smaller than 2-3 mu m, dominate in most open oceanic regions, such as in the Pacific Ocean. In these areas, the dominant carotenoid of photosynthetic eukaryotes is 19’-hexanoyloxyfucoxanthin (19HF), considered to be a diagnostic marker for prymnesiophytes. This suggests that this class could be a major component of eukaryotic picoplankton. despite the fact that virtually no prymnesiophyte has been described to date from this size das ;. To address this question, we assessed prymnesiophyte diversity and abundance in natural picoplankton communities, using a molecular approach. Total genomic DNA was isolated from 3-mu m-filtered samples collected in the Pacific Ocean. Small subunit (18S) ribosomal RNA genes (rDNA) were amplified by the polymerase chain reaction (PCR) using universal eukaryotic primers. The relative abundance of 18S rDNA from prymnesiophytes was quantified using group-specific and eukaryotic 18S rDNA probes. The percentage of the prymnesiophyte versus total 18S rDNA was much lower than the percentage of prymnesiophytes calculated on the basis of pigment analyses for the same samples. 18S rDNA libraries from five samples were screened using a prymnesiophyte-specific oligonucleotide probe, and 14 nearly complete 18S rDNA sequences were retrieved. Phylogenetic analysis of these sequences established the presence of several prymnesiophyte lineages with no equivalent among cultivated species.

Okada Y, Oyama Y, Chikahisa L, Satoh M, Kanemaru K, Sakai H, Noda K (2000) Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes : a flow cytometric study. Toxicol Lett 117 :123-128


Since some of organotins, accumulated in edible mollusks of aquatic environments, exert a variety of toxic actions on experimental animals, it causes concern for the health of humans. We examined the effects of tri-n-butyltin chloride (TBT) and other organotins (triethyltin chloride, trimethyltin chloride, triphenyltin chloride and tetrabutyltin) on cellular content of glutathione (GSH) in rat thymocytes using a flow cytometer to further characterize the toxicity of TBT. When the cells were incubated with TBT at concentrations of 3 nM or more for 15 min, the cellular content of GSH dose-dependently decreased. However, it completely or partly recovered until 180 min even in the continued presence of TBT. This recovery was temperature-sensitive, suggesting an involvement of metabolic process. The efficacy of TBT to decrease the cellular content of GSH was greater than those of other organotins. Results suggest that TBT and some organotins at environmentally relevant (nanomolar) concentrations significantly reduce the cellular content of GSH, suggesting that they increase the vulnerability to some biological and chemical insults.

Olson RJ, Sosik HM, Chekalyuk AM, Shalapyonok A (2000) Effects of iron enrichment on phytoplankton in the Southern Ocean during late summer : active fluorescence and flow cytometric analyses. Deep-Sea Research Part Ii-Topical Studies in Oceanography 47 :3181-3200

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Eight shipboard iron-enrichment experiments were carried out during the late summers of 1997 and 1998 in the Ross Sea and the Polar Front, respectively, as part of the US JGOFS Southern Ocean program. Using active fluorescence techniques (pump-during-probe flow cytometry/microfluorometry and fast repetition rate fluorometry) and flow cytometry, we examined responses of phytoplankton to iron enrichment over time scales of days. Results of both individual cell and bulk water measurements suggest that physiological iron limitation was widespread in the Ross Sea gyre in the late summer, but that in the region just south of the Polar Front other factors were limiting phytoplankton growth. In the five experiments in which responses to enrichment occurred, all the phytoplankton groups we examined, with the exception of cryptophytes, responded to iron enrichment by increasing normalized variable fluorescence (F-v/F-m) over several days. Normalized variable fluorescence of cryptophyte cells was typically higher than that of other cells and often near the maximum observed. Significant correlations were observed between ambient iron concentrations and normalized variable fluorescence at the beginning of each experiment, and also between ambient iron and the response of normalized variable fluorescence to enrichment. These relationships, which have not been previously documented, support the use of ambient active fluorescence measurements to predict iron-limiting conditions without conducting incubations. (C) 2000 Elsevier Science Ltd. All rights reserved.

Parthuisot N, Catala P, Lemarchand K, Baudart J, Lebaron P (2000) Evaluation of ChemChrome V6 for bacterial viability assessment in waters. Journal of Applied Microbiology 89 :370-380

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The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In constrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.

Paul JH, Alfreider A, Kang JB, Stokes RA, Griffin D, Campbell L, Ornolfsdottir E (2000) Form IA rbcL transcripts associated with a low salinity/high chlorophyll plume (’Green River’) in the eastern Gulf of Mexico. Marine Ecology-Progress Series 198 :1-8

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Coastal plumes of low salinity water that extend hundreds of kilometers offshore into oligotrophic waters are often found in the Gulf of Mexico. To characterize one such feature, a series of photoautotrophic activity and biomass parameters were measured at 2 stations in the eastern Gulf of Mexico, including pigments by high performance liquid chromatography (HPLC), autotrophic picoplankton abundance by flow cytometry, photoautotrophic C-14-HCO3- fixation, and Ribulose-1,5-diphosphate carboxylase large subunit gene (rbcL) transcriptional activity. One sampling site (Stn 4) was in a 15 m deep, low salinity (29.8 ppt) plume 242 km west of Tampa Bay. This feature contained relatively high chlorophyll a (chl a) concentrations, carbon fixation rates, and Synechococcus cell abundance (8.6 x 10(4) cells ml(-1)) at 3 m depth and a relatively shallow (45 m depth) subsurface chlorophyll a maximum (SCM). We also found a high level (1.1 ng l(-1)) of Form IA rbcL mRNA in the surface water as determined by probing with a 1.1 kb Synechococcus WH7803 rbcL probe. Form IA rbcLs have been found to occur mainly in chemosynthetic autotrophic bacteria but have recently been described in Synechococcus WH7803 as well as in Prochlorococcus GP2. In contrast, a nearby station outside of the plume (Stn 7) had a SCM at 83 m, lower chi a, Synechococcus cell counts, and carbon fixation rates in the surface waters. The amount of Form IA rbcL was only about 3% of the concentration found in the surface waters of Stn 4. Both stations had an abundance of Prochlorococcus cells (> 10(5) ml(-1)) at intermediate depths (20 to 70 m). The picoeucaryote community occurred principally below the Prochlorococcus community, coinciding with the SCM, and was composed of diatoms, prymnesiophytes, and pelagophytes as determined by HPLC pigment analysis. This report represents the first description of Form IA rbcL transcriptional activity in the marine environment, and indicates that Form IA rbcL-containing picoplankton (like Prochlorococcus GP2 and Synechococcus WH7803) may be important in the primary production of low salinity, surface water plumes of the Gulf of Mexico.

Paul JH, Alfreider A, Wawrik B (2000) Micro- and macrodiversity in rbcL sequences in ambient phytoplankton populations from the southeastern Gulf of Mexico. Marine Ecology-Progress Series 198 :9-18

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Ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO) large subunit genes (rbcL) were obtained by amplification and cloning of 554 or 614 bp sequences of indigenous phytoplankton populations at 2 stations in the southeastern Gulf of Mexico. One station (Stn 4) was located in a low salinity, high chlorophyll plume (the ’Green River’) which has previously been shown to contain elevated levels of Form IA rbcL mRNA while the other (Stn 7) was in oligotrophic, oceanic water. A diversity of rbcL sequences was obtained, spanning 3 of the 4 evolutionary clades of Form I RuBisCOs. Six nucleotide sequences obtained from Stn 4 were closely related (92 to 96% similar) to the Form IA-containing Prochlorococcus GP2. Flow cytometry and pigment analysis indicated that Prochlorococcus was abundant at this site. Other sequences found included a Form IB rbcL closely related to prasino-phytes, and Form ID sequences related to prymnesiophytes, diatoms, and pelagophytes. One sequence was nearly identical to the pelagophyte, Pelagomonas calceolata. At Stn 7, sequences were obtained that were more deeply rooted, and less similar to rbcLs in existing databases (77 to 83 % similar), and no Form IA rbcLs were detected. HPLC pigment signatures and flow cytometry data were consistent with the forms obtained by cloning, The similarity of the 6 Prochlorococcus GP2-like sequences (93 to 98%) is consistent with the phenomenon of molecular microdiversity as found at other loci in marine land other environmental) microorganisms.

Peperzak L, Vrieling EG, Sandee B, Rutten T (2000) Immuno flow cytometry in marine phytoplankton research. Scientia Marina 64 :165-181

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The developments in the combination of flow cytometry and immunology as a tool to identify, count and examine marine phytoplankton cells are reviewed. The concepts of immunology and now cytometry are described. A distinction is made between quantitative and qualitative immunofluorescence. Quantitative immunofluorescence, the identification and enumeration of phytoplankton cells, is the research area that has advanced rapidly in the past decade, and is reviewed extensively. Key steps of quantitative immunofluorescence, fixation and immunolabel intensity, are discussed in more detail. Qualitative immunofluorescence is a new, hardly explored but highly interesting development in which qualitative-physiological-variables related to e.g. nutrient limitation or primary production are measured in individual cells instead of phytoplankton populations as a whole. Several combinations of immunological probes, bath for species identification and for physiological measurements, are proposed. A special case of qualitative immunofluorescence is the measurement of phytoplankton toxins in single cells from natural populations. It is anticipated that the future use of semiconductor nanocrystals or "quantum dots" as fluorophores will greatly enhance signal detection in flow cytometry, and hence in both quantitative and qualitative immunofluorescence applications.

Pettersen EF, Bjerknes R, Wergeland HI (2000) Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry. Fish Shellfish Immunol 10 :695-710


Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.

Pfeiffer CJ, Sharova LV, Gray L (2000) Functional and ultrastructural cell pathology induced by fuel oil in cultured dolphin renal cells. Ecotoxicol Environ Saf 47 :210-217


Investigations were undertaken to elucidate in a marine mammal renal cell culture system the toxicity and some of the mechanisms of cytopathology in a standardized preparation following exposure to No. 1 fuel oil. Cell survivability of a cultured SP1K renal cell line from the Atlantic spotted dolphin Stenella plagiodon was reduced in a dose-dependent manner after a 12-h exposure to fuel oil. Early morphologic changes reflecting cytotoxicity, as revealed by transmission electron microscopy, included enlarged rough endoplasmic reticula, cytoplasmic vacuolization, and degenerative cytoplasmic inclusions, but mitochondria remained resistant. Assessment of extracellular proton loss by microphysiometry of cultured cells revealed fuel oil-induced enhancement of proton loss that was dependent upon both protein kinase C and renal epithelial Na(+)/H(+) counter-transport functioning, as the specific inhibitors H-7 and amiloride reduced this stimulatory petroleum effect. Cell cycle progression and apoptosis (programmed cell death) were studied in dolphin renal cells exposed to fuel oil for 12, 24, and 48 hours. The toxicant increased the percentage of cells in GO/GI phase and decreased the percentage of cells in S phase starting after 24 hours. The number of cells undergoing early apoptosis was also increased after 24 hours.

Radford JL, Hutchinson AE, Burandt M, Raftos DA (2000) Effects of metal-based environmental pollutants on tunicate hemocytes. J Invertebr Pathol 76 :242-248


Tunicates are filter feeding marine invertebrates that are susceptible to environmental contamination by toxic metals and polyaromatic hydrocarbons. Recently, we have shown that tunicate immune reactions are profoundly affected by exposure to tributyltin (TBT) and copper, both of which are components of marine antifouling paints. This study tests the effects of those pollutants on the hemocytes of tunicates. Immunofluorescence labeling with an anti-hemocyte monoclonal antibody demonstrated that the antigenic structure of the circulating hemocyte population was substantially affected by TBT and copper. Antigen-positive hemocytes were also found to accumulate in the pharyngeal papillae of TBT-exposed tunicates. Histological analyses indicated that this cellular accumulation in pharyngeal papillae involved refractile vacuolated hemocytes. Refractile vacuolated cells from TBT-exposed tunicates also occurred at greater frequencies in the circulating hemolymph, and had altered morphologies, compared to cells from nontreated controls. These data confirm that exogenous metals can have profound effects on the hemocytes of tunicates.

Reckermann M (2000) Flow sorting in aquatic ecology. Scientia Marina 64 :235-246

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Flow sorting can be a very helpful tool in revealing phytoplankton and bacterial community structure and elaborating specific physiological parameters of isolated species. Droplet sorting has been the most common technique. Despite the high optical and hydro-dynamic stress for the cells to be sorted, many species grow in culture subsequent to sorting. To date, flow sorting has been applied to post-incubation separation in natural water samples to account for group-specific physiological parameters (radiotracer-uptake rates), to the production of clonal or non-clonal cultures from mixtures, to the isolaton of cell groups from natural assemblages for molecular analyses, and for taxonomic identification of sorted cells by microscopy. The application of cell sorting from natural water samples from the Wadden Sea, including different cryptophytes, cyanobacteria and diatoms, is shown, as well as the establishment of laboratory cultures from field samples. The optional use of a red laser to account for phycocyanine-rich cells is also discussed.

Rohwer F, Azam F (2000) Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling. Appl Environ Microbiol 66 :1001-1006


Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3’-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3’-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3’-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.

Rose JB, Kubba J, Tobutt KR (2000) Chromosome doubling in sterile Syringa vulgaris x S. pinnatifolia hybrids by in vitro culture of nodal explants. Plant Cell Tissue and Organ Culture 63 :127-132

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The aim was to produce tetraploid forms of hybrids of Syringa vulgaris x S. pinnatifolia to restore fertility and enable further breeding to be undertaken. Excised nodal sections of three selections were pre-cultured for 5 days then treated with a range of colchicine concentrations for 1, 2 or 3 days, after which they were washed three times in sterile distilled water and cultured on shoot proliferation medium. Frequent movement to fresh medium was beneficial to survival. Three successive experiments established the range of concentrations of colchicine, 0.05 mM to 0.25 mM, at which tetraploids were likely to be produced. Thus a protocol for the production of tetraploids was established. Cytometric analysis showed that tetraploid forms of two selections were produced.

Sanchez P, Llorente MT, Castano A (2000) Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents : mitomycin C, vincristine sulfate and benzo(a)pyrene. Mutat Res 465 :113-122


The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.

Smit J, Sherwood CS, Turner RFB (2000) Characterization of high density monolayers of the biofilm bacterium Caulobacter crescentus : Evaluating prospects for developing immobilized cell bioreactors. Canadian Journal of Microbiology 46 :339-349

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Caulobacters are biofilm-forming members of the natural flora of soil and aquatic environments, which exhibit several characteristics that make them attractive for development of high surface area microbial bioreactors or biosensors. Although caulobacters are well characterized genetically, little is known about their biofilm-forming characteristics as a monoculture, or their tolerance of bioreactor-like conditions. Here we investigated the ability of caulobacters to spontaneously form high-density monolayers on artificial surfaces under a variety of environmental conditions, using phase contrast image analysis to assess biofilm density, and epifluorescence with the vital stain DiBAC(TM) to assess viability. With adequate nutrition, extremely dense monolayers formed within 24-48 h, and maintained near 100% viability in experiments ranging up to 22 days. When areas were abraded to remove cells, repopulation occurred rapidly with characteristics similar to the population of a clean surface. When established monolayers were starved for nutrients, a significant fraction of the cells detached from the surface, and cells remaining on the surface no longer tested as viable. Within 4-6 h of nutrient restoration, however, cells in the monolayer again appeared normal and tested as 100% viable. This is the first demonstration that Caulobacter crescentus is stable and amenable to high density monolayer growth and resists starvation, though some cells may express a programmed response to detach from the surface under severe nutrient limitation.

Suzuki MT, Taylor LT, DeLong EF (2000) Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5’-nuclease assays. Appl Environ Microbiol 66 :4605-4614


Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5’-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5’-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5’-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.

Talyzina NM, Moldowan JM, Johannisson A, Fago FJ (2000) Affinities of Early Cambrian acritarchs studied by using microscopy, fluorescence flow cytometry and biomarkers. Review of Palaeobotany and Palynology 108 :37-53

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Examination and chemical analysis of extremely well-preserved microfossils from the Lower Cambrian Lukati Formation in Estonia suggests that acritarchs from among the genera Globosphaeridium, Skiagia, Comasphaeridium and Lophosphaeridium have dinoflagellate affinities. The investigation presents a combination of transmitted light microscopy, fluorescence microscopy and flow cytometry, and biomarker analysis that demonstrates a new method for the investigation of problematic organic-walled microfossils. For the chemical analysis, Lukati Formation acritarchs were separated from prasinophycean tasmanitids by size and then divided into two fractions in accordance with the intensity of their autofluorescence signal. Biomarker molecules were generated by pyrolysis directly from isolated acritarch organic walls and studied using gas chromatography-mass spectrometry-mass spectrometry (GC-MS-MS) and metastable reaction monitoring (MRM)-GC-MS. The analysis supported previously made suggestions that acritarchs include microorganisms of different biological affinities. All acritarch fractions contain the common steranes (cholestane, 24-methylcholestane and 24-ethylcholestane) that are characteristic molecules for eukaryotes. However, the dinoflagellate-related biomarkers, dinosterane and 4 alpha-methyl-24-ethylcholestane, were concentrated only in the fraction containing highly autofluorescent acritarchs. Additional chemical analyses of microfossils from the Lower Cambrian Buen Formation of North Greenland confirmed the presence of the dinoflagellate-related biomarkers at a second Early Cambrian locality. (C) 2000 Elsevier Science B.V. All rights reserved.

Tanaka Y, Yamaguchi N, Nasu M (2000) Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry. J Appl Microbiol 88 :228-236


The enzymatic activity and viability of Escherichia coli O157:H7 in natural river water was determined by flow cytometry. River water was collected at two sites (an agricultural area and an industrial area) on the Aigawa River (Osaka, Japan). To facilitate estimation of the physiology of E. coli O157 in natural river water, bacterial cells in the water were stained with 6-carboxyfluorescein diacetate (6CFDA) and propidium iodide (PI). The cells were sorted into two populations, using a flow cytometer, based on their esterase activity. Each population was stained with E. coli O157:H7 fluorescent antibody (FA), and E. coli O157:H7 cells were observed in the esterase-active population. River water samples collected at the same points were incubated with yeast extract containing antibiotics to prevent cell division, and bacterial cells in the incubated samples were stained with PI and FA. Escherichia coli O157:H7 existed in both the viable (elongated and/or fattened) and inactive bacterial population determined by flow cytometry. These results indicate that E. coli O157:H7 may retain metabolic activity and growth potential in the natural aquatic environment.

Uysal Z (2000) Pigments, size and distribution of Synechococcus spp. in the Black Sea. Journal of Marine Systems 24 :313-326

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Pigments, size and distribution of Phycoerythrin-containing unicellular cyanobacteria Synechococcus spp. within the euphotic zone were studied for the first time in April-May 1994 in the western and southwestern Black Sea by epifluorescence microscopy and flow-cytometry. Synechococcus was present in varying quantities at every station and depth studied. Surface spatial distribution of Synechococcus revealed that cells were much more abundant in offshore waters than near coastal regions under the direct influence of the Danube river. Minimum and maximum cell concentrations ranged between 9 x 10(2) and 1.45 x 10(5) cells/ml at the surface, between 2 x 10(3) and 1.23 10(5) cells/ml at the chlorophyll sub-maximum layer, and between 1.3 x 10(2) and 3.5 x 10(2) at the nitrite maximum layer. Cells at the chlorophyll sub-maximum layer (based on in-situ fluorometer readings) fluoresce brighter and longer than the ones at the surface and lower depths. Spectral properties of chromophore pigment types of total 64 clonal isolates from different depths down to the lower layer of the euphotic zone (similar to 60 m) in the southern Black Sea coast revealed that all have type 2 phycoerythrobilin in common, lacking in phycourobilin. In vivo fluorescence emission maxima for the phycoerythrobilin were about the same (similar to 578 nm) for all isolates. All isolates examined showed in vivo absorption maxima at between 435 and 442 nm and at about 681 nm due to chlorophyll-a. Based on the flow cytometer mean forward light scatter data for size distribution, it could be concluded that cells at the surface mixed layer (0-10 m) were larger in cell size than the cells at lower depths (20-60 m). (C) 2000 Elsevier Science B.V. All rights reserved.

Veldhuis MJW, Kraay GW (2000) Application of flow cytometry in marine phytoplankton research : current applications and future perspectives. Scientia Marina 64 :121-134

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A brief overview is given of current applications of flow cytometry (FCM) in marine phytoplankton research. This paper presents a selection of highlights and various technical and analytical problems we encountered during the past 10 years. In particular, the conversion of the relative values obtained in terms of size and fluorescence applying FCM to quantitative estimates of cell size, pigment concentration, genome size etc., is addressed. The introduction of DNA-cell-cycle analysis made easily assessable by flow cytometry has been of great importance, allowing in situ measurement of species specific growth rates. Key questions in ecology such as factors determining the wax and wane of phytoplankton bloom can now be better answered in terms of species specific growth and mortality. Finally, flow cytometry provides derailed information of the physiological status of the individual algal cells. New staining methods enable us to distinguish between viable and non-viable cells and so will help us to elucidate the importance of "automortality" in aquatic ecosystems.

Verity PG, Williams SC, Hong Y (2000) Formation, degradation, and mass : volume ratios of detritus derived from decaying phytoplankton. Marine Ecology-Progress Series 207 :53-68

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The mass of non-living particulate organic matter, or detritus, often exceeds that of living plankton in pelagic environments ; in coastal waters the difference can be 10-fold. Relatively little is known about the dynamics of this pool of organic matter because it has not been previously possible to accurately determine its magnitude. A recent approach utilizing fluorescence microscopy (Verity et al. 1996) provided estimates of the volume of detritus. Here, laboratory experiments were conducted to estimate carbon:volume (C:V) and nitrogen:volume (N:V) conversion ratios of detritus formed by 2 phytoplankton species when incubated in darkness in the presence of bacteria. The volume of detritus was measured directly, along with total particulate organic carbon (POC) and nitrogen (PON), and that contained in the associated phytoplankton and bacterial communities. Detrital carbon and nitrogen were estimated by difference and compared to detrital volume, from which conversion factors were calculated. The physiological state of the bacteria was assessed using a recently developed fluorescent stain and molecular probe protocol (Williams et al. 19983. The 2 phytoplankton cultures were degraded at different rates. At the times of peak bacterial abundances, most of the remaining bulk POC and PON was in the form of bacteria cells. Conversion efficiencies, however were only 8 to 9 % (carbon) and 10 to 11% (nitrogen). The fraction of the bacterial community composed of active cells was inversely related to the C:N ratio of the bulk particulate matter in both cultures, although with different absolute values. C:N ratios of detritus, distinct from surrounding bacteria and phytoplankton, were typically 35 to 50 but varied during the 56 d incubations because bacteria selectively degraded PON compared to POC. C:V and N:V ratios were typically 0.09 to 0.11 and 0.002 to 0.004 pg mum(-3) in fresh detritus, respectively, and ratios declined as the detritus degraded in the presence of bacteria. Mean C:V and N:V ratios were 0.05 to 0.11 and 0.0014 to 0.0031, respectively. These ratios indicate that detritus derived from phytoplankton cultures contains reduced densities of organic carbon and nitrogen compared to living plankton. They provide the means to directly estimate the carbon and nitrogen content of natural detritus, although the C:V and N:V ratios of cultures need to be compared to those estimated from natural plankton communities.

Vinogradov AE (2000) Larger genomes for molluskan land pioneers. Genome 43 :211-212

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The terrestrial pulmonate mollusks were found to have the significantly larger genomes than the aquatic pulmonates. Being shown in the independent phylogenetic branch, this phenomenon suggests that the previously observed genome enlargement in the vertebrate land pioneers (amphibians and lungfishes) was not casual. As in the vertebrates, the larger molluskan genomes are also more GC-rich.

Vives-Rego J, Lebaron P, Nebe-von Caron G (2000) Current and future applications of flow cytometry in aquatic microbiology. FEMS Microbiol Rev 24 :429-448


Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems : (i) its tremendous velocity to obtain and process data ; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis ; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are : cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Vives-Rego J, Lopez-Amoros R, Guindulain T, Garcia MT, Comas J, Sanchez-Leal J (2000) Microbial aspects of linear alkylbenzene sulfonate degradation in coastal water. Journal of Surfactants and Detergents 3 :303-308

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Viable and total bacteria were determined during linear alkylbenzene sulfonate (LAS) degradation in coastal seawater. Viable bacteria were determined by plate counts on marine agar media, while total bacteria were determined by flow cytometry after SYTO-13 staining. LAS degradation was monitored by high-performance liquid chromatography analysis. Seawater samples with LAS at 5 mg/L, incubated in the laboratory (20 degrees C, 100 rpm, 30 d), showed in most cases a similar evolution in bacterioplankton abundance over time, characterized by three phases : (i) a progressive increase in bacterial density ; (ii) a later decrease ; and (iii) a fluctuating stationary phase. Bacterioplankton degraded the LAS by growing to populations with a high percentage of viable bacteria. The bacteria were rapidly grazed by protozoa, preventing anomalous high bacterial growth and ensuring the later channeling of LAS carbon to upper trophic levels.

Worden AZ, Chisholm SW, Binder BJ (2000) In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with RRNA-targeted peptide nucleic acid probes. Appl Environ Microbiol 66 :284-289


A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.

Yang H, Zhang F, Guo X (2000) Triploid and Tetraploid Zhikong Scallop, Chlamys farreri Jones et Preston, Produced by Inhibiting Polar Body I. Mar Biotechnol (NY) 2 :466-475


Triploid scallops are valuable for aquaculture because of their enlarged adductor muscle, and tetraploids are important for the commercial production of triploids. We tested tetraploid induction in the zhikong scallop by inhibiting polar body I in newly fertilized eggs. The ploidy of resultant embryos was determined by chromosome counting at 2- to 4-cell stage and by flow cytometry thereafter. Embryos from the control groups were mostly diploids (79%), along with some aneuploids. Embryos from the treated groups were 13% diploids, 18% triploids, 26% tetraploids, 13% pentaploids, and 36% aneuploids. Tetraploids, pentaploids, and most aneuploids suffered heavy mortality during the first week and became undetectable among the larvae at day 14. Five tetraploids (2%) were found among a sample of 267 spat from one of the replicates, and none was detected at day 450. The adductor muscle of triploid scallops was 44% heavier (P <.01) than that of diploids, confirming the value of the triploid technology in this species.

Yokomaku D, Yamaguchi N, Nasu R (2000) Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments. Applied and Environmental Microbiology 66 :5544-5548

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A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are Lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.

Zubkov MV, Sleigh MA, Burkill PH, Leakey RJG (2000) Bacterial growth and grazing loss in contrasting areas of North and South Atlantic. Journal of Plankton Research 22 :685-711

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Samples were collected from the top 200 m of the water column at 50 stations during two cruises in different, near equinoctial seasons on an Atlantic transect near the 20 degrees W meridian between 50 degrees N and 50 degrees S. These samples were analysed to determine characteristics of the heterotrophic bacterial populations. Flow cytometry was used to enumerate these bacteria and determine their average size so as to calculate their biomass. Heterotrophic bacterial production, and the rate of grazing of these bacteria by heterotrophic nanoplankton in the main depth layers, were determined using H-3 thymidine and C-14 leucine techniques. The biomass of heterotrophic nanoplankton in these layers was determined using a glucosaminidase assay. Five provinces were distinguished along the transect and characterized by average values of all measured parameters. The relative composition and activity of the microbial community in the water columns within each province changed little between the two cruises. Lowest heterotrophic bacterial biomass of 1-2 mg C m(-3) and production of 0.1-0.2 mg C m(-3) day(-1) were found in the northern and southern Atlantic gyres, and were relatively similar in both seasons. Biomass and production were 2-4 times higher in the northern and southern temperate waters, and in equatorial waters, than in the gyres and tended to show more seasonal variation. Production and biomass in the layer below the pycnocline were lower by 10-30% and about 50%, respectively, than values determined in the surface mixed layer, and varied less with latitude. Depth-integrated values of these two parameters were generally of similar size in the mixed water layer and the layer of the chlorophyll maximum and pycnocline, and tended to vary with season. The specific growth rate of heterotrophic bacteria was in the range 0.05 to 0.12 day(-1) in the top mixed layer at all latitudes. In spite of the elevated temperatures, bacterial growth appears to be restricted by a shortage of nutrients so that the microbial community cycles very slowly, with a turnover time of the order of 1 week or more. The depth-integrated biomass of heterotrophic nanoplankton was generally about 100% of the heterotrophic bacterial biomass in the same water. Grazing by these nanoplankton at the rate measured could consume all of the new production of heterotrophic bacteria in all waters, and they probably control the populations of both heterotrophic and phototrophic bacteria.

Zubkov MV, Sleigh MA, Burkill PH, Leakey RJG (2000) Picoplankton community structure on the Atlantic Meridional Transect : a comparison between seasons. Progress in Oceanography 45 :369-386

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Samples collected from 10 depths at 25 stations in September-October 1996 and 12 depths at 28 stations in April-May 1997 on an Atlantic Meridional Transect between the British Isles and the Falklnnd Islands were analysed by flow cytometry to determine the numbers and biomass of four categories of picoplankton : Prochlorococcus Spp, Synechococcus Spp, picoeuckaryotic phytoplankton and heterotrophic bacteria. The composition of the picoplankton communities confirmed earlier findings (Zubkov, Sleigh, Tarran, Burkill & Leakey, 1998) about distinctive regions along the transect and indicated that the stations should be grouped into five provinces : northern temperate, northern Atlantic gyre, equatorial, southern Atlantic gyre and southern temperate, with an intrusion of upwelling water off the coast of Mauritania between the northern Atlantic Eyre and equatorial waters. Prochlorococcus was the most numerous phototrophic organism in waters of both northern and southern gyres and in the equatorial region, at concentrations in excess of 0.1x10(6)ml(-1) ; it also dominated plant biomass ill the gyres, but the biomass of the larger picoeukaryotic algae equalled that of Prochlorococcus in the equatorial region, higher standing stocks of both Prochlorococcus and picoeukaryotes were present in spring than in autumn in waters of both gyres. In temperate waters at both ends of the transect the numbers and biomass of picoeukaryotes and, more locally, of Synechococcus increased, and the Synechococcus, particularly, were more numerous in spring than in autumn. There was a pronounced southward shift of the main populations of both Synechococcus and Prochlorococcus in April-May in comparison to those of September-October, associated with seasonal changes in solar radiation, the abundance of Prochlorococcus dropping sharply near the 17 degrees C contour, while Synechococcus was still present at temperatures below 10 degrees C. Picoeukaryotes were more tolerant of low temperatures and lower light levels. often being more abundant in samples from greater depths, where they contributed to the deep chlorophyll maximum. Heterotrophic bacterial numbers : and biomass tended to be highest in those samples where phototrophic biomass was greatest, with peaks in temperate and equatorial waters, which were shifted southwards in April-May compared with September-October. (C) 2000 Elsevier Science Ltd. All rights reserved.