2005

mardi 21 avril 2009
par   G. Grégori

Bec B, Husseini-Ratrema J, Collos Y, Souchu P, Vaquer A (2005) Phytoplankton seasonal dynamics in a Mediterranean coastal lagoon : emphasis on the picoeukaryote community. Journal of Plankton Research 27 :881-894

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The dynamics of the phytoplankton community were investigated in a marine coastal lagoon (Thau, NW Mediterranean) from February 1999 to January 2000. Dilution experiments, chlorophyll a (Chl a) size-fractionation and primary production measurements were conducted monthly. Maximum growth and microzooplankton grazing rates were estimated from Chl a biomass fractions to separate pico- from nano- and microphytoplankton and by flow cytometry to distinguish between picoeukaryotes and picocyanobacteria. In spring, the phytoplankton community was dominated by Chaetoceros sp. and Skeletonema costatum, which represented most of biomass (B) and primary production (P). Nano- and microphytoplankton growth was controlled by nutrient availability and exceeded losses due to microzooplankton grazing (g). Picoeukaryote and cyanobacteria growth was positively correlated with water temperature and/or irradiance, reaching maximum values in the summer (2.38 and 1.44 day(-1) for picoeukaryotes and cyanobacteria, respectively). Picophytoplankton accounted for 57% of the biomass-specific primary productivity (P/B). Picophytoplankton was strongly controlled by protist grazers (g = 0.09-1.66 day(-1) for picoeukaryotes, g = 0.25-1.17 day(-1) for cyanobacteria), and microzooplankton consumption removed 71% of the daily picoplanktonic growth. Picoeukaryotes, which numerically dominate the picoplankton community, are an important source of organic carbon for the protistan community and contribute to the carbon flow to higher trophic levels.


Biegala IC, Cuttle M, Mary I, Zubkov M (2005) Hybridisation of picoeukaryotes by eubacterial probes is widespread in the marine environment. Aquatic Microbial Ecology 41 :293-297

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Most general and group-specific eubacterial probes hybridised picoeukaryotes in coastal waters (Brittany, France) and cultures of the dominant picoeukaryotes from this environment (Micromonas pusilla and Pelagomonas calceolata). This is either because they matched the 16S rRNA from organelles or because of the presence of symbiotic or antagonist intracellular bacteria. The general eubacterial probe (EUB338) hybridised 84% of the picoeukaryotes, while the group-specific probes hybridised 3, 16, 10 and 34% of the picoeukaryotes for cyanobacteria (CYA664), alpha-proteobacteria (ALF968), gamma-proteobacteria (GAM42a) and Cytopbaga-Flavo-Bacteria (CF319), respectively. The results show that the hybridisation of eukaryote 16S rRNA by prokaryote probes can lead to significant errors in prokaryote counts, in particular for less well-represented groups such as cyanobacteria, with errors of 17% in the studied sample. In addition, we revealed for the first time at this scale that up to 44% of the picoeukaryotes contained intracellular prokaryotes. This finding might have serious implications for understanding the functioning of the microbial loop. Finally, because SSU rRNA databases have significantly been extended in recent years, we showed that the probe PLA886, which targets Planctomycete 16S rRNA, labelled 87%. of the picoeukaryotes by hybridising their 18S rRNA. Consequently, the design of this probe should be refined for future studies, and the presence of similar changes in probe specificity should be checked regularly when using hybridisation-based techniques.


Camacho FG, Belarbi EH, Garcia MCC, Miron AS, Chile T, Chisti Y, Grima EM (2005) Shear effects on suspended marine sponge cells. Biochemical Engineering Journal 26 :115-121

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Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Muller protocol [W.E.G. Muller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge : primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205-219]. Supplementation of the standard Ca2+ and Mg2+-free artificial seawater of the Muller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75 rpm essentially killed all the viable cells within 2.5 h, but no loss of viability occurred at a higher agitation speed of 100 rpm for up to 6 It when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3 degrees C instead of the normal 17 degrees C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within 10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell-cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact). (c) 2005 Elsevier B.V. All rights reserved.


Campbell L, Carpenter EJ, Montoya JP, Kustka AB, Capone DG (2005) Picoplankton community structure within and outside a Trichodesmium bloom in the southwestern Pacific Ocean. Vie Et Milieu-Life and Environment 55 :185-195

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Phytoplankton composition and community structure in the southwestern Pacific Ocean were examined at sea using flow cytometry and epifluorescence microscopy to explore the relationships among distributions of picophytoplankton populations and a variety of nitrogen fixing cyanobacteria. The cruise track began in New Zealand, extended north via New Caledonia to 13 degrees S, and continued east to 13.9 degrees S 173.2 degrees W. The track crossed a large bloom of the filamentous nitrogen-fixing cyanobacteria Trichodesmium centered around New Caledonia. Within Trichodesmium blooms, abundances of Synechococcus were elevated 10-fold ; however, there was no significant enrichment of Prochlorococcus, picoeukaryotic algae, or heterotrophic bacteria. Unicellular coccoid cyanobacteria (> 2 mu m), which resemble the nitrogen-fixing Crocosphaera spp., were observed in waters > 27 degrees C along the eastward track and were most abundant at the deep oceanic stations where Trichodesmium was absent or present at very low abundances. These Crocosphaera-rich, Trichodesmium-poor stations were characterized by lower dissolved iron concentrations compared to coastal stations where Trichodesmium tended to be more abundant. Given the apparent mutually exclusive distributions of these two groups of cyanobacteria, further examination of N-2 fixation within the pico- and nanoplankton components of the phytoplankton community is needed.


Casotti R, Mazza S, Brunet C, Vantrepotte V, Ianora A, Miralto A (2005) Growth inhibition and toxicity of the diatom aldehyde 2-trans, 4-trans-decadienal on Thalassiosira weissflogii (Bacillariophyceae). Journal of Phycology 41 :7-20

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A common aldehyde present in marine and freshwater diatoms, 2-trans, 4-trans-decadienal (A3), is involved in the wound-activated response of diatoms to copepod grazing. Upon breakage of the diatom cell membrane, aldehydes are enzymatically produced by the rapid conversion of precursors and strongly impact copepod reproduction by impairing egg production and hatching success, inducing teratogenic embryos modifications. In this study, A3 was assayed with the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle. The aldehyde concentration necessary to reduce 50% growth rate (EC50) was 0.29 mg.L-1. Decadienal was found to inhibit T. weissflogii cell growth in a dose- and time-dependent manner, with irreversible effects after 24 h of exposure. Decadienal induced a degenerative process, through modifications of cell membrane characteristics, interference with cell cycle progression, and with cell metabolic activity, leading to cell death. A preferential action of A3 on dividing cells was observed. Photosynthetic efficiency significantly decreased upon exposure to the aldehyde, paralleled by an increase in diatoxanthin, suggesting a protective role of this xanthophyll, usually involved in photoprotection. Dying cells exhibited the morphological and biochemical features that bear close resemblance to apoptosis of mammalian cells, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. These data are the first direct evidence to show aldehydes are toxic to diatoms. We suggest a possible nontoxic role of such compounds as chemical signals of unfavorable conditions within the phytoplankton communities, which may be relevant for the population dynamics of diatoms during blooms.


Christaki U, Vazquez-Dominguez E, Courties C, Lebaron P (2005) Grazing impact of different heterotrophic nanoflagellates on eukaryotic (Ostreococcus tauri) and prokaryotic picoautotrophs (Prochlorococcus and Synechococcus). Environ Microbiol 7 :1200-1210

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Autotrophic picoplankton (<3 microm) composed of both prokaryotes and eukaryotes are the most abundant primary producers on Earth. In this study we examined the ingestion of the picoeukaryote Ostreococcus tauri by different marine heterotrophic nanoflagellates (HNF) with various morphologies, swimming and feeding behaviours. Cultures of specific bacterivorous nanoflagellates (Rhynchomonas nasuta, Jakoba libera, and a culture of Cafeteria sp./Monosiga sp.) and natural nanoflagellate populations were used as grazers. For comparison with Ostreococcus, we used similar-sized prokaryotes as prey, Prochlorococcus and Synechococcus. We observed large species-specific differences in terms of : use of picoautotrophs among nanoflagellates, time lag between prey addition and prey consumption (0-196 h), grazing rate (0-0.12 h(-1)), growth rate (0-0.3 h(-1)) and maximum abundance of HNF reached in experimental bottles (e.g. from 10(4) to 10(5) cells ml(-1), for a natural coastal population and a Cafeteria sp./Monosiga sp. culture feeding Ostreococcus respectively). Overall, this study shows that the nanoflagellate community composition is conclusive for picoautotrophic community structure and, vice versa, the picoautotrophic community structure favours or inhibits the growth of some nanoflagellate groups.


Corzo A, Rodriguez-Galvez S, Lubian L, Sobrino C, Sangra P, Martinez A (2005) Antarctic marine bacterioplankton subpopulations discriminated by their apparent content of nucleic acids differ in their response to ecological factors. Polar Biology 29 :27-39

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Bacterial abundances determined in Drake Passage and Bransfield and Gerlache Straits (Antarctica) in the Austral summer ranged from 0.78 to 9.4x10(5) cells ml(-1), and were positively correlated with standing stocks of Chl a. Two bacterial subpopulations were discriminated based in their different levels of green fluorescence and wide angle light scatter (SSC) per cell after SYTO-13 staining for the first time in Antarctic waters. High nucleic acid (HNA) and low nucleic acid (LNA) subpopulations differed considerably in their response to changes in environmental variables. The apparent content of nucleic acids per cell for the HNA subpopulation (FL1-HNA) showed vertical profiles similar to those of Chl a, including the presence of a maximum at the subsurface chlorophyll maximum. FL1-HNA was positively correlated with Chl a. No similar trends were observed for the LNA fraction. HNA and LNA subpopulations differed in the response of the wide angle light scatter signal to environmental factors as well. SSC-HNA decreased strongly with depth and was positively correlated with Chl a. Again, no similar trends were observed for the LNA subpopulation. The percentage of HNA cells (%HNA) ranged between 35.0 and 76.7% and showed a general tendency to increase with depth. This increase seemed to be larger when the stratification of the water column was higher. Differences in grazing pressure could be responsible of the unexpected vertical distribution of HNA cells. Our results shows that in situ LNA and HNA bacterioplankton subpopulations are under different ecological controls and likely to play different trophodynamic roles in Antarctic waters.


Cottrell MT, Waidner LA, Yu LY, Kirchman DL (2005) Bacterial diversity of metagenomic and PCR libraries from the Delaware River. Environmental Microbiology 7 :1883-1895

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To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization ( FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups ( e. g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.


Devilla RA, Brown MT, Donkin M, Readman JW (2005) The effects of a PSII inhibitor on phytoplankton community structure as assessed by HPLC pigment analyses, microscopy and flow cytometry. Aquat Toxicol 71 :25-38

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Measurements of the stress imposed by a PSII inhibiting herbicide (Irgarol 1051) on the composition of a phytoplankton community was investigated by comparing chemotaxonomy, as determined by high performance liquid chromatography (HPLC), optical microscopy and analytical flow cytometry (AFC). Changes in community structure were induced in microcosms containing a natural marine phytoplankton community exposed to different concentrations of Irgarol 1051 (0.5 and 1.0 microgl-1). Microcosms were maintained under controlled laboratory conditions in semi-continuous culture over 120 h. Class-specific phytoplankton biomass (chlorophyll a) was estimated using CHEMTAX analyses of pigment concentrations. Microscopic identification and carbon content estimates were cross-correlated with CHEMTAX and also with AFC enumeration/size classifications of major phytoplankton groups. CHEMTAX-HPLC analyses and microscopy results demonstrated that prasinophytes and prymnesiophytes were the most affected groups following exposure to Irgarol 1051. The selective reductions in both classes as estimated by both techniques revealed similar trends. Results for chlorophytes and dinoflagellates showed these groups to be most tolerant to Irgarol 1051. Indeed, class-specific biomass for chlorophytes as determined by CHEMTAX and microscopy were correlated (R2=0.53) which demonstrated an increase in both abundance and carbon content following exposures to Irgarol 1051. Abundances of nanoeukaryotes as determined by microscopy afforded good agreement with results from AFC (R2=0.8), although for picoeukaryotes, abundances were underestimated by microscopy (R2=0.43). The relative performance of the selected techniques is discussed.


Devilla RA, Brown MT, Donkin M, Tarran GA, Aiken J, Readman JW (2005) Impact of antifouling booster biocides on single microalgal species and on a natural marine phytoplankton community. Marine Ecology-Progress Series 286 :1-12

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Phytoplankton were exposed to 4 antifouling booster biocides (Sea-Nine 211(R), Irgarol 1051(R), diuron and zinc pyrithione) to investigate toxicological responses. Initially, single species/single biocide exposure experiments revealed changes in pigment ratios under all biocide exposures for the prymnesiophyte Emiliania huxleyi, but not for the cyanophyte Synechococcus sp. Growth inhibition results following 72 h exposures indicated that Synechococcus sp. was more tolerant to zinc pyrithione (NOEC of 1.0 mug 1(-1)) and Sea-Nine 211(R) (NOEC of 0.9 mug 1(-1)) than E. huxleyi (EC50 of 0.54 and EC50 of 0.35 mug 1(-1), respectively). In contrast, Synechococcus sp. was more sensitive to diuron (EC50 of 0.55 mug 1(-1)) than E. huxleyi (EC50 of 2.26 mug 1(-1)), whereas exposure to Irgarol 1051(R) similarly impacted both species (EC50 of 0.16 and 0.25 mug 1(-1), respectively). In addition, the impact on photosynthesis and on pigment chernotaxonomy was investigated through a laboratory exposure experiment using a natural phytoplankton community. Pigment signatures were measured by High Performance Liquid Chromatography (HPLC) and densities of size-classified phytoplankton groups were monitored using Analytical Flow Cytometry (AFC). Group-specific sensitivity of the natural phytoplankton community was detected through pigment composition after 72 h exposure to 5 mug 1(-1) zinc pyrithione and 10 mug 1(-1) Sea-Nine 211(R). Zeaxanthin increased proportionally, indicating a relative increase in Cyanophyceae. This result was corroborated using AFC. Primary production, estimated by C-14-HCO3- uptake, was compared to maximum quantum yield of Photosystem II (F-V/F-M), which was quantified by Fast Repetition Rate Fluorimetry (FRRF). The 2 techniques were in good agreement (R-2 = 0.89, p = 0.0001), both primary production and F-V/F-M being impaired by exposure to all biocides tested. These results are discussed in the context of the potential environmental impact of biocides on phytoplankton communities and the ecological implications of any modifications in species composition.


Devos L, Clymans K, Boon N, Verstraete W (2005) Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples. J Appl Microbiol 99 :916-925

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AIMS : To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS : The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS : The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY : This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).


Diaz MR, Fell JW (2005) Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex. J Clin Microbiol 43 :3662-3672

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Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 10(1) to 10(3) genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.


Ellison CK, Burton RS (2005) Application of bead array technology to community dynamics of marine phytoplankton. Marine Ecology-Progress Series 288 :75-85

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Studies of the dynamics of marine microplanktonic systems have been hampered by a lack of high-throughput technologies for the simultaneous identification and quantification of the many taxa comprising such communities. To address this problem, we describe a DNA-hybridization-based method of analysis employing bead array technology. Species- and/or genus-specific probes for 8 phytoplankton taxa were designed and applied to the microplankton community sampled from coastal waters off La Jolla, California, over an exploratory time series 10 d in length. Bulk DNA extractions from 1 l of seawater were used without PCR amplification. Taxa analyzed comprised 4 dinoflagellates, 3 diatoms, and 1 coccolithophorid. The results demonstrated that specifically targeted oligonucleotide probes can be used in this fashion with a high degree of binding-specificity, dependent on hybridization temperature, and that standard curves relative to target-cell concentration can be constructed. The use of 2 different probes for each taxon can provide added confidence that probes are taxon-specific. Further, single-species assays and multiplexed assays were generally in good agreement, as were assays of replicate seawater samples. Once sets of probes are developed for particular groups of taxa, the bead array system appears to provide a technological platform with great promise for high-throughput analyses of microplanktonic communities.


Engelmann P, Palinkas L, Cooper EL, Nemeth P (2005) Monoclonal antibodies identify four distinct annelid leukocyte markers. Developmental and Comparative Immunology 29 :599-614

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This paper describes for the first time the production and characterization of a library of monoclonal antibodies (anti-EFCC clones) raised against coelomocyte (leukocyte) markers of Eisenia fetida earthworm. Leukocyte subgroups are components of earthworm innate immunity that require a more precise characterization using immunological markers. Flow cytometry, immunocytochemistry, and immunoprecipitation analyzed and confirmed the specificity of anti-EFCC clones. Anti-EFCC mAbs revealed different leukocyte subpopulations and various staining patterns on tissues. Two functional assays (e.g. phagocytosis and encapsulation) further characterized EFCC clusters revealing a common coelomocyte marker and three subpopulation-specific markers. No crossreactivity was found on human, mouse, rat or cells from Drosophila melanogaster but immunoreactivity was detected on snail (Planorbarius corneus) tissues. Immunohistochemical results suggest mesodermal origin of all coelomocyte subgroups that agree with classical morphological analyses. (c) 2004 Elsevier Ltd. All rights reserved.


Eschbach E, John U, Reckermann M, Cembella AD, Edvardsen B, Medlin LK (2005) Cell cycle dependent expression of toxicity by the ichthyotoxic prymnesiophyte Chrysochromulina polylepis. Aquatic Microbial Ecology 39 :85-95

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The coupling of toxicity expression with cell-cycle phases was studied in the toxic marine prymnesiophyte Chrysochromulina polylepis Manton & Parke, Clone B1511. Cell synchronisation of cultures in exponential or early stationary growth phases under nutrient-replete conditions was achieved by manipulation of the photoperiod. Chlorophyll a (chl a) and cell number increased in a stepwise manner, but were asynchronous, with chl a increasing during the light period and cell number increasing during the dark period. In the course of the light period, nearly all cells clustered in the G1 (Gap 1) phase, which lasted for about 20 h. DNA synthesis (S phase) occurred mainly in the dark during a discrete period (about 4 h) and G2 (Gap 2) and mitosis (M) were always completed before the end of the dark period. Toxicity expression, measured by the erythrocyte lysis assay (ELA), exhibited a dramatic drop in LC50 values (increase in toxicity) during the light period, although this effect was less pronounced after the first 2 generations of cell division when the cultures had entered the stationary phase, Similarly, haemolytic activity per unit cell volume decreased by a factor of 3 to 4 during the dark period over the first 48 h, but became irregular towards the end of the experiment. In this study, the light-dependent effect on toxicity and relationship to discrete phases of the cell cycle are demonstrated for the first time in a prymnesiophyte.


Fan Y, Hu J, Li J, Yang Z, Xin X, Wang J, Ding J, Geng M (2005) Effect of acidic oligosaccharide sugar chain on scopolamine-induced memory impairment in rats and its related mechanisms. Neurosci Lett 374 :222-226

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In this study we evaluated the effect of a novel, marine-derived, acidic oligosaccharide on scopolamine-induced amnesia in rats using the Morris water maze test. The results show that 30-day administration of this oligosaccharide, referred to as acidic oligosaccharide sugar chain (AOSC), to rats attenuates memory impairment by scopolamine, as evaluated by shortened escape latency, swimming distance, and increased swimming time of rats with memory impairment induced by scopolamine in the quadrant where the platform is placed. The data additionally suggest that an appropriate dose of scopolamine, a traditional muscarinic receptor antagonist, elevates oxidative damage in brain, characterized by inactivation of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and consequently, inhibition of ATPase in the hippocampus and cerebral cortex. AOSC ameliorates oxidative injuries caused by scopolamine by increasing the activities of SOD, GSH-Px, and ATPase. Further investigation by flow cytometry revealed that AOSC significantly reduces the overloading of intracellular free calcium ion ([Ca2+]i), thus suppressing apoptosis induced by H2O2 in human neuroblastoma SH-SY5Y cells. These findings suggest that AOSC can induce cognitive improvement via its antioxidant activity.


Fischer UR, Kirschner AKT, Velimirov B (2005) Optimization of extraction and estimation of viruses in silty freshwater sediments. Aquatic Microbial Ecology 40 :207-216

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The present study focused on the optimization of procedures for the extraction of viruses from silty freshwater sediments for subsequent enumeration. Viral abundance in 2 different shallow backwater systems of the River Danube (Austria) ranged from 1.45 x 10(9) to 9.58 x 10(9) particles ml(-1) wet sediment. The highest virus yields from the bulk of the sediments were obtained by 1 min sonication (3 x 20 s intervals, with 10 s interruptions). An increase in sonication time of up to 5 min decreased viral counts by an average of 15 %. Since dissolved DNA within sediment samples could bind to the nucleic acid stain and thereby inflate viral estimates, sediment samples are often treated with DNase before the staining procedure. Moreover, they are usually centrifuged and diluted to a high extent in order to avoid interference of particulate material with virus counting. Centrifugation led to a reduction of viral numbers by 2 to 36 % compared to untreated samples and did not reduce the background fluorescence ; thus counting of viruses was not facilitated. Diluting 2000 x with Milli-Q water always provided an average of 19 % lower viral numbers than diluting 4000 x. Treatment with DNase had no significant effect on virus counting, with viral numbers in untreated samples being on average 96 % of those in DNase-containing samples. Additionally, 2 different nucleic acid stains were compared-viruses stained with SYBR Gold fluoresced brighter than those stained with SYBR Green I and fluorescence lasted longer, while background fluorescence was reduced sufficiently, thus facilitating virus counting. Viral numbers using SYBR Gold were on average twice of those obtained with SYBR Green I. The mean efficiency of virus extraction was 88.8% using the protocol outlined in this paper, and was thus slightly higher than that obtained in previous sediment investigations.


Fuchs BM, Woebken D, Zubkov MV, Burkill P, Amann R (2005) Molecular identification of picoplankton populations in contrasting waters of the Arabian Sea. Aquatic Microbial Ecology 39 :145-157

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The composition of picoplankton in the southern oligotrophic, northern mesotrophic waters and deep oxygen minimum zone (OMZ) of the Arabian Sea was determined by 16S ribosomal RNA gene cloning and fluorescence in situ hybridisation (FISH). It was hypothesised that the composition of the heterotrophic picoplankton would be different in these contrasting waters. To reduce the total diversity, cells were sorted by flow cytometry according to their scatter and DNA content before PCR amplification. The 16S rRNA clone libraries resulting from flow-sorted populations were different and often dominated by a small number of clades. Libraries from the Prochlorococcus-dominated southerly waters were dominated by sequences related to uncultured clusters of SAR11, SAR86 and Actinobacteria (HGC 1). From surface waters of the Synechococcus-dominated northern part of the Arabian Sea, mostly sequences related to the uncultured gammaproteobacterial group ’Svalbard’ and HGC I were retrieved. The clone libraries from the OMZ were also dominated by sequences falling in the clades SARI 1 and SAR406, but included sequences related to those of sulfate-reducing (Desulfosarcina, Desulfofrigus) and sulfide-oxidising bacteria (endosymbionts of Riftia and Calyptogena). With a recently developed more sensitive FISH protocol approximately 60% of all DAPI stained cells could be identified by general probes as Bacteria, Cren- or Euryarchaeota in both provinces of the Arabian Sea ; 40% remained undetected. On this level and on that of the major phylogenetic groups like Alpha- and Gammaproteobacteria only minor differences were detected by FISH. However, the composition of heterotrophic picoplankton clearly differed for the proteobacterial subgroups SAR86, SAR11 and SAR116. These were more abundant in the oligotrophic waters throughout the water column than in the mesotrophic surface waters and the OMZ. This supports our original hypothesis that the contrasting waters in the Arabian Sea harbor different heterotrophic picoplankton communities. In the future, FISH with a larger set of probes for more narrow phylogenetic groups will enable us to quantify these differences in more detail.


Gin KYH, Neo SY (2005) Microbial populations in tropical reservoirs using flow cytometry. Journal of Environmental Engineering-Asce 131 :1187-1193

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Flow cytometry was applied in the study of bacteria and phytoplankton populations in five tropical reservoirs. Water quality between different reservoirs was compared and correlation analyses were carried out to investigate how the biomass of bacteria and phytoplankton related to other water quality parameters measured (i.e., temperature, dissolved oxygen, pH, conductivity, water transparency, turbidity, chlorophyll a, and total nitrogen and phosphorus). Average chlorophyll a concentrations were typically greater than 20 mu g/L. Bacteria populations detected with flow cytometry were generally small in size (typically <0.08 mu m(3) or 0.3 mu m equivalent spherical diameter) and contributed less than 13% of the total microbial biomass. Subpopulations of pico-, ultra-, and net phytoplankton were discriminated flow cytometrically by their red and orange autofluorescence. Cyanobacteria dominated four out of the five reservoirs in terms of numbers but only contributed more than 50% of the microbial biomass in two of the reservoirs. In general, local reservoirs were found to be phosphorus limited and alkaline conditions favored the growth of phytoplankton and bacteria.


Goddard VJ, Baker AC, Davy JE, Adams DG, De Ville MM, Thackeray SJ, Maberly SC, Wilson WH (2005) Temporal distribution of viruses, bacteria and phytoplankton throughout the water column in a freshwater hypereutrophic lake. Aquatic Microbial Ecology 39 :211-223

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Analytical flow cytometry (AFC) and denaturing gradient gel electrophoresis (DGGE) were used to investigate the diversity and dynamics of viruses, bacteria and phytoplankton communities in a hypereutrophic freshwater lake. Samples were taken from different depths throughout the water column over an annual cycle. Priest Pot is a small lake in the Lake District in NW England (UK), and has been well characterised in previous studies ; however, little is known of the diversity and dynamics of the virus community. Virus abundance was shown to change over both spatial and temporal scales, and appeared to be closely linked to other biotic and abiotic parameters. The highest virus concentrations occurred in the deepest part of the lake at a depth of 3.2 m, in the anoxic layer, at the same time as a peak of abundance of green sulphur bacteria. Sequence analysis of a DGGE band that occurred at the same time suggested a bacterium similar to Chlorobium spp., a green sulphur bacterium, comprised part of this bacterial bloom.


Gordon R, Parkinson J (2005) Potential roles for diatomists in nanotechnology. Journal of Nanoscience and Nanotechnology 5 :35-40

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Diatoms produce diverse three-dimensional structures that, due to their exponential rate of growth, may be of use in the manufacture of components for nanotechnology as an alternative to present linear lithographic techniques. Vapor replacement of the silicon permits the conversion of diatom silica valves and other structures to metal/ceramics, with no loss of structure. The literature on diatom nanotechnology is reviewed, along with suggestions on how diatomists might enhance this emerging technology. There is a need for a systematics based catalog of parts (via genomics technologies), improved diatom culture techniques, better understanding of the mechanisms of diatom morphogenesis and motility, and genetic manipulation, mutagenesis, and selection, as via the chemostat-like compustat. Given the self-motility of raphid diatoms, they could form the basis for industrially useful nanobots.


Groben R, Medlin L (2005) In situ hybridization of phytoplankton using fluorescently labeled rRNA probes. Methods Enzymol 395 :299-310

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Phytoplankton are one of the major components of ecosystem processes and play an important role in many biogeochemical cycles in the marine and freshwater environment. Despite their importance, many microalgae are poorly described and little is known of broad spatial and temporal scale trends in their abundance and distribution. Reasons for this are that microalgae are often small, lack distinct morphological features, and are unculturable, which make analyses difficult. It is now possible by using molecular biological techniques to advance our knowledge of aquatic biodiversity and to understand how biodiversity supports ecosystem structure, dynamics, and resilience. We present in this chapter a brief review of the progress that has been made in analyzing microalgae from populations to the species level. The described methods range from DNA fingerprinting techniques, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs), to microsatellites, which are used in population studies, to sequence analysis, which help to reconstruct the evolutionary history of organisms and to examine relationships at various taxonomic levels. Special emphasis is given to the application of molecular probes for the identification and characterization of microalgal taxa. The fast and secure identification of phytoplankton, especially of toxic species, is important from an ecological and economical point of view and whole-cell hybridization with specific fluorochrome-labeled probes followed by fluorescence microscopy or flow cytometry offers a fast method for this purpose. In this context, we present a detailed protocol for fluorescence in situ hybridization (FISH) of ribosomal RNA (rRNA) probes that can be applied to many algal cell types and discuss practical considerations of its use.


Guo PY, Zhu YM, Zhang ZJ (2005) Flow cytometric analysis of fine particles in a eutrophic lake. Luminescence 20 :135-137

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Fine particles play an important role, not only in aquatic biogeochemical processing but also in the distribution, transfer and transformation of pollutants in the aquatic environment. Flow cytometry, widely used in biomedical research, allows fast counting and optical analysis of individual particles. Organic autotrophic particles contain naturally fluorescing pigments, such as chlorophyll and phycoerythrin. Different populations have different sizes and pigments. They also have different ratios of pigments. In general, side angle scatter (SSC) is related to the size, shape and refractive index of particles. When a 488 nm wavelength was used to excite chlorophyll and phycoerythrin fluorescence, the pigments of organic autotrophic particles emitted red and orange light. Fine particles were detected by flow cytometry (FCM) in the southern part of a eutrophic lake in winter. We found that organic autotrophic particles belonged to three populations, which represented only 15.89% of total fine particles. Organic non-living particles and inorganic particles represented the greater part (84.11%) of total fine particles. This study also demonstrated that flow cytometry is well suited to the dynamic monitoring and analysis of natural water aquatic particles that were difficult to study with traditional methods.


Helton RR, Cottrell MT, Kirchman DL, Wommack KE (2005) Evaluation of incubation-based methods for estimating virioplankton production in estuaries. Aquatic Microbial Ecology 41 :209-219

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Assessment of the role of viral lysis in aquatic microbial communities requires a robust means of estimating viral production (VP). Here, 3 incubation-based VP methods (fluorescently labeled viruses [FLV], dilution [DIL], and thymidine incorporation [TdR]) were evaluated in water samples from the Delaware and Chesapeake Bays. In Chesapeake Bay samples, average VP rates were 10-fold higher for FLV and DIL (similar to 3 to 25 x 10(10) viruses l(-1) d(-1)) than for TdR (similar to 0.2 to 3 x 10(10) viruses l(-1) d(-1)). Estimates of viral-mediated bacterial mortality (VMM) indicate that FLV overestimates VP in eutrophic waters, since > 100 % of bacterial production (BP) would have been consumed through viral lysis. DIL and TdR VP methods gave more realistic estimates of VMM with respect to BP. The FLV method provides estimates of both VP and virus removal rates ; however, it requires preparation of a viral tracer and twice as many microscopic enumerations as the DIL method. DIL was the least difficult and most efficient method ; however, bacterial loss during vacuum diafiltration resulted in poor replicability. TdR gave similar VP estimates to DIL, but requires use of a large and poorly constrained conversion factor. With methodological improvements in bacterial cell recovery, DIL should be the most widely applicable method for estimation of VP in highly productive estuarine waters.


Houdan A, Probert I, Van Lenning K, Lefebvre S (2005) Comparison of photosynthetic responses in diploid and haploid life-cycle phases of Emiliania huxleyi (Prymnesiophyceae). Marine Ecology-Progress Series 292 :139-146

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Emiliania huxleyi is a ubiquitous coccolithophore, capable of forming large blooms. This species presents a digenetic heteromorphic life cycle, the non-motile diploid phase typically bearing coccoliths and the flagellated haploid phase being non-calcified. Oxygen production rates at different irradiances of both phases were studied in mid-exponential and transitional growth phases in cultures grown under identical conditions. There were no significant differences in basic photosynthetic parameters (alpha(chl a), maximum light utilization coefficient ; p,hi, the light-saturated maximal rate of max(’) photosynthesis ; Ek, the light-saturation parameter) between the 2 life-cycle phases ; however, whereas the diploid phase did not exhibit photoinhibition at irradiances up to 1000 mu mol photons m(-2) s(-1), photoinhibition was recorded in the haploid phase above 400 to 500 mu mol photons M-2 s-1 and photosynthetic rate decreased to ca. 75 % of P-max at 1400 mu mol photons m(-2) s(-1). The 2 phases cultured under identical (non-saturating in terms of light) conditions did not present any significant differences in pigment content. These results are discussed in an ecological context. The lack of photoinhibition could confer a competitive advantage on the diploid stage, notably in a turbulent environment. For the haploid stage, the occurrence of photoinhibition may indicate niche separation (spatial and/or temporal) relative to the diploid phase.


Jacquet S, Domaizon I, Personnic S, Ram ASP, Hedal M, Duhamal S, Sime-Ngando T (2005) Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France). Freshwater Biology 50 :627-645

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1. We performed three, 1-week in situ experiments in March-April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus- induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM).
2. A dilution approach, applied to pretreated grazer-free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal.
3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally.
4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7%(expt 1), 3.4%(expt 2) and 5.7%(expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3%(expt 1), 3.7%(expt 2) and 6.3%(expt 3).
5. It is clear that the dilution-FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer.


Jing X, Wenbin Z (2005) Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri). Fish Shellfish Immunol 19 :17-25

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Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively ; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.


Jochem FJ (2005) Short-term physiologic effects of mechanical flow sorting and the Becton-Dickinson cell concentrator in cultures of the marine phytoflagellata Emiliania huxleyi and Micromonas pusilla. Cytometry A 65 :77-83

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BACKGROUND : In contrast to large, high-efficiency cytometers, mechanically sorting benchtop instruments provide a feasible alternative for shipboard cell sorting of oceanic microbial communities. However, sorting efficiency of these instruments is constrained by their maximum sorting rate of approximately 300 cells/s and by constant dilution of sorted samples by sheath flow. These factors often render too low sorted cell concentrations for postsorting experiments of oceanic phytoplankton populations of low natural abundance. A Cell Concentrator module has been marketed to overcome these dilution effects. Postsorting experiments also have to consider potential physiologic effects of cell sorting. Short-term physiologic effects on phytoplankton photosynthetic rates and esterase activities by mechanical flow sorting and cell concentration and on the efficiency of the Cell Concentrator module are evaluated. METHODS : Increasing numbers of the oceanic phytoflagellates Micromonas pusilla and Emiliania huxleyi were sorted and concentrated, and recovery in the concentrated samples was compared with the sorted-only samples (concentration rate) and the total number of sorted cells (recovery rate). Photosynthetic rates and metabolic activities of sorted and sorted/concentrated cells were compared with unsorted cells. Photosynthetic rates were estimated from 14CO2 uptake experiments and metabolic activity quantified cytometrically after cleavage of fluorescein diacetate. RESULTS : Irrespective of the total number of sorted cells, concentration rates between concentrated and sorted cells remained mostly below 10-fold and did not increase with the number of concentrated cells. Recovery rates in the concentrated samples amounted to fewer than 10% of total sorted cells, except for forceful resuspension attempts in the Concentrator insert (25-44%), which might be unsuitable for delicate species. Cell sorting resulted in a 24-49% decrease in photosynthetic rates. Metabolic activity within metabolically active cells was not affected by cell sorting, but the share of metabolically active cells decreased by 32-37%. Cell concentration did not affect metabolic activity or the fraction of active cells but did increase photosynthetic rate several-fold compared with unsorted cells. CONCLUSION : Low recovery of concentrated cells, probably due to cell adhesion to the filer bottom of the Concentrator insert, render the Cell Concentrator of limited use to overcome dilution problems of mechanical flow sorting, particularly when results are extrapolated to natural, low-abundance populations. Severe changes in photosynthetic rates also render concentrated cells suspicious for subsequent physiologic experiments. Mechanical sorting alone also exhibited significant physiologic effects on sorted cells, some of which might not be temporary. Comparable effects between mechanical sorting and droplet sorting as previously reported confirm that physiologic effects might be caused predominantly by shear stress and laser exposure during cytometric analysis rather than the sorting process. Sufficient recovery time must be allowed before postsorting experiments, but potential changes in cell physiology from the natural conditions during postsorting recovery must be considered.


Katano T, Kaneda A, Takeoka H, Nakano S (2005) Seasonal changes in the abundance and composition of picophytoplankton in relation to the occurrence of ’Kyucho’ and bottom intrusion in Uchiumi Bay, Japan. Marine Ecology-Progress Series 298 :59-67

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Uchiumi Bay experiences intermittent physical events of ’Kyucho’ and bottom intrusion. A Kyucho is an intrusion of warm surface water from the Kuroshio in the Pacific Ocean. Bottom intrusion, which contains a large amount of nitrates, phosphates, and silicates, slips through just above the continental shelf. We investigated seasonal changes in the abundance of Prochlorococcus, Synechococcus, and eukaryotic picophytoplankton while monitoring Kyucho and bottom intrusion from March to October 2002. Kyucho and bottom intrusion frequently occurred from June to September. Relatively high concentrations of nitrate + nitrite (> 0.8 mu mol N l(-1)) and phosphate (> 0.1 mu mol P l(-1)) were found when bottom intrusion occurred. The cell densities of Prochlorococcus were relatively high (> 1 X 10(4) cells ml(-1)) when Kyucho occurred. Those of Synechococcus were high (2 to 30 x 10(4) Cells ml-1) during the period of thermal stratification except in July, when bottom intrusion occurred. The cell densities of eukaryotic picophytoplankton were high (2 to 8 x 10(4) cells ml(-1)) in May and July. To examine the effects on picophytoplankton growth of the nutrients supplied by bottom intrusion, we conducted nutrient-enrichment experiments. The growth rates of Prochlorococcus and Synechococcus were not stimulated by the addition of any kinds of nutrients. The growth rates of Prochlorococcus were negative in most cases. In July, the growth rate of eukaryotic picophytoplankton was stimulated by nitrate and phosphate additions. Thus, Prochlorococcus detected in Uchiumi Bay might have been transported by Kyucho from the Pacific Ocean and could therefore not grow vigorously. Synechococcus may have been flushed out by bottom intrusion, and its growth was not limited by the nutrient concentrations. Eukaryotic picophytoplankton was abundant in spring, and its growth might have been limited by the nutrient concentrations in some cases. These results suggest that Kyucho and bottom intrusion have different effects on the abundance and growth rate of the 3 picophytoplankton groups.


Lin J, Yan XJ, Zheng L, Ma HH, Chen HM (2005) Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites. J Appl Microbiol 99 :1373-1382

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AIMS : To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS : After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS : Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY : Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.


Lombardi VR, Fernandez-Novoa L, Etcheverria I, Seoane S, Cacabelos R (2005) Effects of fish-derived lipoprotein extracts on activation markers, Fas expression and apoptosis in peripheral blood lymphocytes. Int Immunopharmacol 5 :253-262

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Several factors may influence numbers and function of peripheral blood lymphocytes (PBLs) by different processes. We conducted this study to evaluate the effect of E-CAB-94011 and E-JUR-94013, two marine fish extracts from S. scombrus and T. trachurus, respectively, on in vitro PBLs activation and on the expression and functionality of Fas, a cell surface molecule that plays a central role in immune homeostasis and cytotoxic activity. PBLs from 24 healthy volunteers were isolated and flow cytometry was performed to measure the state of activation, Fas expression and apoptosis of PBLs. Functionality of Fas was tested by assessing apoptosis after incubation of isolated lymphocytes with agonistic anti-Fas antibodies in blood samples treated with both E-CAB-94011 and E-JUR-94013. Studies on the lymphocyte cell marker suggest a clear immune activation as measured by the increased levels of CD25, CD8, CD38, CD19 and HLA-DR in vitro expression on lymphocytes treated with both extracts. In addition, a significant reduction in the percentages of apoptotic CD19(+)CD38(+) double positive lymphocytes could be demonstrated in the treated samples with respect to controls (p<0.05). Therefore the present results indicate that both E-CAB-94011 and E-JUR-94013 in vitro are powerful immunoregulatory, increasing immune surveillance.


Longnecker K, Sherr BF, Sherr EB (2005) Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem. Applied and Environmental Microbiology 71 :7737-7749

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We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [H-3] leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.


Lund ED, Soudant P, Chu FL, Harvey E, Bolton S, Flowers A (2005) Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus. Dis Aquat Organ 67 :217-224

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Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.


Martin AP, Zubkov MV, Burkill PH, Holland RJ (2005) Extreme spatial variability in marine picoplankton and its consequences for interpreting Eulerian time-series. Biol Lett 1 :366-369

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A high-resolution mesoscale spatial survey of picoplankton in the Celtic Sea, using flow cytometry, reveals cell concentrations of Synechococcus spp. cyanobacteria and heterotrophic bacteria that vary up to 50-fold over distances as short as 12 km. Furthermore, the range of abundances is comparable to that typically found on seasonal scales at a single location. Advection of such spatial variability through a time-series site would therefore constitute a major source of ’error’. Consequently, attempts to model and to investigate the ecology of these globally important organisms in situ must take into account and quantify the hitherto ignored local spatial variability as a matter of necessity.


Matson CW, Palatnikov G, Islamzadeh A, McDonald TJ, Autenrieth RL, Donnelly KC, Bickham JW (2005) Chromosomal damage in two species of aquatic turtles (Emys orbicularis and Mauremys caspica) inhabiting contaminated sites in Azerbaijan. Ecotoxicology 14 :513-525

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The Caspian region and specifically the Apsheron peninsula of Azerbaijan are known to be polluted with a variety of environmental contaminants. These complex mixtures of contaminants make risk assessment difficult. We used the flow cytometry method (FCM) and the micronucleus assay (MN) to assess chromosomal damage in aquatic turtles (Emys orbicularis, the European pond turtle ; and Mauremys caspica, the Caspian turtle) inhabiting contaminated wetlands in Azerbaijan. Evidence of genetic damage was found for two sites, Neftchala and Sumgayit, relative to a reference site, Ali Bairamly. Sediment samples from each site were analyzed for PAHs and mercury to evaluate potential contaminant associations with genetic damage. A significant positive correlation was documented between three-ring PAH sediment concentrations and FCM estimates of chromosomal damage in E. orbicularis. These data combine to show that the contaminated wetlands in Sumgayit and Neftchala are genotoxic and that three-ring PAHs are likely a significant influence on observed genotoxicity.


Miao B, Li J, Fu X, Ding J, Geng M (2005) T-cell receptor (TCR)/CD3 is involved in sulfated polymannuroguluronate (SPMG)-induced T lymphocyte activation. Int Immunopharmacol 5 :1171-1182

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Sulfated polymannuroguluronate (SPMG) has entered the Phase II clinical trial as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate in China. Proliferation assays showed that SPMG was effective at enhancing the proliferative response of T lymphocytes either with or without concanavalin A (ConA) stimulation. Flow cytometry (FCM) and fluorescence microscope examination revealed the significant binding of SPMG to T lymphocytes. The significant engagement of SPMG with TCR/CD3 complex was verified by competitive inhibition assay and one of the SPMG binding proteins purified by affinity chromatography from thymocyte membrane preparations was further confirmed to be CD3 component of TCR/CD3 complex via Western blotting analysis. In addition, SPMG was demonstrated to dramatically interact with ConA in a multivalent manner by surface plasmon resonance (SPR) assay. Notably, the concomitant presence of ConA and SPMG facilitated each other’s binding to T cells. Together, the simultaneous interactions of SPMG with TCR/CD3 and with ConA can be highly proposed to facilitate the cross-linking of these molecules, and thus favoring costimulatory signaling, which serves to well explain the immunopotentiation and anti-human immunodeficiency virus (HIV) activities of SPMG.


Misumi I, Vella AT, Leong JA, Nakanishi T, Schreck CB (2005) p,p’-DDE depresses the immune competence of chinook salmon (Oncorhynchus tshawytscha) leukocytes. Fish Shellfish Immunol 19 :97-114

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p,p’-DDE, the main metabolite of DDT, is still detected in aquatic environments throughout the world. Here, the effects and mechanisms by which p,p’-DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha) was studied. Isolated salmon splenic and pronephric leukocytes were incubated with different concentrations of p,p’-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p’-DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p’-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leukocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p’-DDE. The effect of p,p’-DDE on leukocytes appeared to vary between developmental stages or seasonal differences. The mitogenic response of leukocytes of chinook salmon exposed to p,p’-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p’-DDE had a significantly lower percentage of Ig+ blasting cells than controls, although the response was biphasic. These results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression.


Miteva VI, Brenchley JE (2005) Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core. Applied and Environmental Microbiology 71 :7806-7818

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The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (< 0.1 mu m(3)) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5 degrees C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-mu m, 0.2-mu m, and even 0.1-mu m filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-mu m-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria ; Cytophaga-Flavobacteria-Bacteroides ; high-G+C gram-positive ; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.


Mou X, Moran MA, Stepanauskas R, Gonzalez JM, Hodson RE (2005) Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations. Appl Environ Microbiol 71 :1405-1416

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15746343

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 microM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, alpha-Proteobacteria) ; Caulobacter (alpha-Proteobacteria) ; and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.


Mou XZ, Moran MA, Stepanauskas R, Gonzalez JM, Hodson RE (2005) Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations. Applied and Environmental Microbiology 71 :1405-1416

<Go to ISI> ://000227702900036

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethyl sulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 mu M DMSP and tracked community shifts with How cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, alpha-Proteobacteria) ; Caulobacter (alpha-Proteobacteria) ; and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.


Ning XR, Li WKW, Cai YM, Liu CG, Shi JX (2005) Standing stock and community structure of photosynthetic picoplankton in the northern South China Sea. Acta Oceanologica Sinica 24 :57-76

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The investigation on community structure of standing stock of photosynthetic picoplankton : Synechococcus (Syn), Prochlorococcus (Pro) and Picoeukaryotes (Euk) and their environmental regulation mechanisms in the northern South China Sea was carried out in the summer of 1999. The results showed that the average abundances of Syn, Pro and Euk in the study sea areas were (5.0 +/- 7.6)x 10(4) cell/cm(3)(51%), (4.6 +/- 4.2)x10(4) cell/cm(3) (47%) and (1.8 +/- 1.1)x10(3) cell/cm(3) (2%) respectively, and those of their carbon biomass were (12.5 +/- 18.9) mu g/dm(3)(74%), (2.7 +/- 2.5) mu g/dm(3)(16%) and (1.7 +/- 1.0) mu g/dm(3) (10%). The most of high values of Syn appeared in the estuaries, coastal zone and continental shelf in the sea area to the east of Leizhou Peninsula and Hainan Island where nutrients were rich, and those appeared in the Beibu. Gulf were the second, while those appeared in continental slope and open sea were tens times lower than the above those. Its distribution in water column was mainly above the thermocline and its abundance below it sharply decreased. Two different populations of Pro were found, the surface population and deep one. The distribution pattern of the former was similar to that of Syn ; while with marked difference from that of the former, the abundance and biomass of the latter markedly increased towards outer sea, continental slope and open sea where nutrients were poor ; the high values in the water column mainly appeared at the bottom of euphotic zone and above the nitrocline, where it often vigorously grow. The distribution difference of Euk in the various sea areas is not as obvious as those of Syn and Pro, but it was high in coastal and shelf waters and low in continental slope and open sea. The high values in the water column were mostly appeared at the bottom of euphotic zone. This difference of distribution pattern for the three type of photosynthetic picoplankton depends on environmental effects and their ecophysiological differences.


Nishimura Y, Kim C, Nagata T (2005) Vertical and seasonal variations of bacterioplankton subgroups with different nucleic acid contents : possible regulation by phosphorus. Applied and Environmental Microbiology 71 :5828-5836

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We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups : bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87 ; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop.


Not F, Massana R, Latasa M, Marie D, Colson C, Eikrem W, Pedros-Alio C, Vaulot D, Simon N (2005) Late summer community composition and abundance of photosynthetic picoeukaryotes in Norwegian and Barents Seas. Limnology and Oceanography 50 :1677-1686

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We investigated marine picoeukaryotic diversity (cells < 3 mu m) in samples collected in late summer 2002 at the boundary between the Norwegian, Greenland, and Barents Seas. The two main Arctic and Atlantic water masses in this region are separated by the polar front. We combined total counts of picoeukaryotes assemblages by flow cytometry and epifluorescence microscopy with taxa detection by tyramide signal amplification-fluorescent in situ hybridization (TSA-FISH) and high performance liquid chromatography (HPLC) pigment analyses. The picoeukaryotic community was primarily composed of photoautotrophs (75% of the cells on average). Members of the division Chlorophyta, in particular the species Micromonas pusilla (Butcher) Manton and Parke, were the major components in truly Arctic waters (32% of the picoeukaryotes, maximum 3,200 cells ml(-1)). M. pusilla was also well represented in coastal waters and at the polar front (25 % of the picoeukaryotes, maximum 9, 100 cells ml(-1)). Haptophyta were prominent in more typical Atlantic waters (up to 35% of the picoeukaryotes, maximum 4,500 cells ml(-1)). Quantification of haptophyte biomass by HPLC pigment analyses and CHEMTAX, and haptophyte abundances by TSA-FISH were in good agreement. This confirms previous studies, which suggested that M. pusilla is a dominant contributor of picoeukaryotic communities in both coastal and nutrient rich environments, whereas haptophytes seem to be more important in open seawaters.


Ortmann AC, Suttle CA (2005) High abundances of viruses in a deep-sea hydrothermal vent system indicates viral mediated microbial mortality. Deep-Sea Research Part I-Oceanographic Research Papers 52 :1515-1527

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Little is known about the distribution and abundance of viruses at deep-sea hydrothermal vents. Based on estimates made using epifluorescence microscopy and the dye YoPro-1, much higher viral abundances were observed at active hydrothermal vents than in the surrounding deep sea. This indicates that viral production was occurring and that viruses were a source of microbial mortality. Samples collected from three actively venting sites (Clam Bed, S&M and Salut) within the Endeavour Ridge system off the west coast of North America had viral abundances ranging from 1.45 x 10(5) to 9.90 x 10(7) ml(-1), while the abundances of prokaryotes ranged from 1.30 x 10(5) to 4.46 x 10(6) ml(-1). The abundances of viruses and prokaryotes in samples collected along the neutrally buoyant plume associated with the Main Endeavour Field were lower than at actively venting sites, with a mean of 5.3 x 10(5) prokaryotes ml(-1) (s.d. 2.9 x 10(5), n = 64) and 3.50 x 10(6) viruses ml(-1) (s.d. 1.89 x 10(6), n = 64), but were higher than non-plume samples (2.7 x 10(5) prokaryotes ml(-1), s.d. 5.0 x 10(4), n = 15 and 2.94 x 10(6) viruses ml(-1), s.d. 1.08 x 10(6), n = 15). Prokaryotic and viral abundances in non-hydrothermal regions were as much as 10-fold higher than found in previous studies, in which sample fixation likely resulted in underestimates. This suggests that viral infection may be a greater source of prokaryotic mortality throughout the deep sea than previously recognized. Overall, our results indicate that virus-mediated mortality of prokaryotes at these hydrothermal-vent environments is significant and may reduce energy flow to higher trophic levels. (c) 2005 Elsevier Ltd. All rights reserved.


Osuchowski MF, Sharma RP (2005) Fumonisin B-1 induces necrotic cell death in BV-2 cells and murine cultured astrocytes and is antiproliferative in BV-2 cells while N2A cells and primary cortical neurons are resistant. Neurotoxicology 26 :981-992

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Fumonisin B-1 (FB1), a mycotoxin produced by Fusarium verticillioides, causes equine leukoencephalomalacia, impairs myelination, and inhibits neuronal growth in vitro. Intact mice do not show brain damage after systemic administration of FBI. We recently reported that intracerebroventricular administration of FBI in mice caused neurodegeneration in the cortex and activation of astrocytes in the hippocampal area ; results suggested that the neuronal damage may be secondary, to activation of immunocompetent non-neuronal cells. Current study investigated effects of FB1 upon murine inicroglial (BV-2) and neuroblastoma (N2A) cell lines, and primary astrocytes and cortical neurons. BV-2 and N2A cultures and cells prepared from neonatal and postnatal brains of BALB/c mice were exposed to various concentrations of FBI for 4 (BV-2 and N2A) or 4 and 8 (astrocytes and cortical neurons) days. FBI at 25 P M decreased viability in BV-2 cells, whereas at 50 mu M caused necrotic but not apoptotic cell death in both BV-2 and primary astrocytes (at day 8 only), assessed by lactic dehydrogenase release, and pripidium iodide and annexin V staining. Thymidine incorporation indicated that 2.5 mu M FBI decreased proliferation in BV-2 cells. DNA analysis by flow cytometry showed that the inhibition was not caused by cell cycle arrest. The mitochondrial activity decreased dose-dependently in BV-2 cells and was significantly elevated at 25 mu M FBI, but not at 50 mu M at days 4 or 8 in astrocytes. In BV-2 cells and primary astrocytes, the expression of TNF alpha and IL-1 beta analyzed by real-time polymerase chain reaction was downregulated at 6 or 24 h. In all cell types tested the FB1 treatment caused accumulation of free sphinganine and decrease in free sphingosine levels at selected time points. Results indicated that primary and established marine brain immunocompetent cells are vulnerable to the FBI-dependent cytotoxicity in vitro whereas neuronal cells are not. The toxic effects at ? the neuronal tissue may therefore be secondary to modulation of astrocyte or glial cell function. (c) 2005 Elsevier Inc. All rights reserved.


Pan LA, Zhang LH, Zhang J, Gasol JM, Chao M (2005) On-board flow cytometric observation of picoplankton community structure in the East China Sea during the fall of different years. Fems Microbiology Ecology 52 :243-253

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On-board flow cytometric determinations of picoplankton abundance (i.e. Synechococcus spp., Prochlorococcus spp., picoeukaryotes and also heterotrophic bacteria) were obtained in the East China Sea in fall of 2000 and 2003. The average abundances of Synechococcus, Prochlorococcus, picoeukaryotes and heterotrophic bacteria were 10(5), 10(5), 10(4) and 10(6) cells ml(-1), respectively. Synechococcus, picoeukaryotes and heterotrophic bacteria were abundant at all the stations and presented higher concentration in the inner shelf where influences from the Changjiang effluent plumes and the coastal upwelling were evident, while Prochlorococcus was absent from the near-shore stations and became the dominant picophytoplankton population in offshore waters, where its abundance was comparable to that for heterotrophic bacteria. All picoplankton groups showed a reduction in cell number with depth, and a positive correlation with water temperature were observed, which reflected the importance of light and temperature on picoplankton growth. A negative relationship with salinity was found for heterotrophic bacteria along two sections across the East China Sea Shelf, and distribution of picoplankton was dominated by different water masses. The fixation could lead to loss in Prochlorococcus cell numbers within one month, and all the picoplankton numbers decreased dramatically after three months. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.


Phe MH, Dossot M, Guilloteau H, Block JC (2005) Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria. Water Research 39 :3618-3628

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Flow cytometry (FCM), combined with staining using two fluorochromes (propidium iodide, PI, or SYBR Green 11 RNA gel stain, SYBR-II), was used to assess nucleic acid injuries to chlorinated drinking water bacteria. Highly fluorescent SYBR-II-stained bacteria were converted to bacteria with low fluorescence after chlorination. PI staining of bacteria exposed to different doses of chlorine showed membrane permeabilisation ([Cl-2] < 0.2 mg L-1) and nucleic acid damage at higher doses ([Cl-2]> 0.3 mgL(-1)). Above a threshold dose (between 1.5 and 3mg Cl2L-1), nucleic acids appeared severely damaged and incapable of being stained by PI or SYBR-II. These results constitute evidence that FCM is a promising too] for assessing drinking water bacteria injuries and for controlling chlorine disinfection efficiency much more rapidly than the standard sensitive but time-consuming heterotrophic plate count method. (c) 2005 Elsevier Ltd. All rights reserved.


Pirker H, Pausz C, Stoderegger KE, Herndl GJ (2005) Simultaneous measurement of metabolic activity and membrane integrity in marine bacterioplankton determined by confocal laser-scanning microscopy. Aquatic Microbial Ecology 39 :225-233

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We simultaneously assessed the metabolic activity and viability of individual bacterioplankton cells in the coastal and open North Sea. Three different techniques were applied to determine cell features related to the physiological status of the cell. SYBR Green I was used to estimate the nucleic acid content of the cell. Propidium iodide (PI) stains cells with a compromised cell membrane, commonly interpreted as indicative of dead cells. Microautoradiography (MA) with radiolabeled glucose and leucine was applied to indicate metabolically active cells. The relative abundance of metabolically active cells determined by MA was usually <20 % of the total abundance of bacteria. In contrast, the percentage of PI-positive cells in the total bacterial community was generally high (similar to 80 %). However, the overwhelming majority (97 %) of cells taking up glucose and leucine were also PI-positive. Apparently, the uptake of radiolabeled substrate is related to PI accumulation in cells, indicating that PI is not a reliable stain to indicate non-active or dead bacteria. We suggest that several methods should be combined to assess the physiological status of individual cells in natural bacterioplankton communities.


Powell LM, Bowman JP, Skerratt JH, Franzmann PD, Burton HR (2005) Ecology of a novel Synechococcus clade occurring in dense populations in saline Antarctic lakes. Marine Ecology-Progress Series 291 :65-80

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The seasonal distribution and abundance of Synechococcus-like morphotypes was investigated in meromictic lakes and coastal areas of the Vestfold Hills, Antarctica. Populations were monitored by flow cytometry utilising phycoerythrin content and small cell size to distinguish the cells from other phytoplankton. In Ace Lake, the Synechococcus bloom commenced in September at the water temperature minimum and peaked in late November. Populations (up to 8 x 10(6) cells ml(-1)) were maximally stratified at a depth of 11 m, corresponding to waters which were supersaturated with oxygen, high in phosphate and which received > 5 pmol photons m(-2) s(-1) of light. At this depth, salinity (30 g kg(-1)) was constant throughout the year and temperature ranged from 4.5 degrees C in October to 10.5 degrees C in February. In late November, high numbers of Synechococcus cells also occurred in the moderate salinity water bodies Lake Abraxas and Pendant Lake (salinity 16.5 to 31.0 g kg(-1)), with populations highly stratified in Lake Abraxas (up to 1.5 x 10(7) cells ml(-1), temperature 8.0 degrees C, salinity 20.3 g kg(-1)) but less so in the colder waters of Pendant Lake (max. 1.5 x 10(7) cells ml(-1), temperature 0.1 to 1.1 degrees C, salinity 31.0 g kg(-1)). Synechococcus populations did not occur in brackish, coastal marine or hypersaline water bodies in the Vestfold Hills. Populations appear to be controlled primarily by temperature and to a lesser extent by light availability and grazing. Characterization of non-axenic cultures indicated that the Antarctic lake Synechococcus populations were similar to other polar picocyanobacteria in terms of cardinal growth temperatures (minimum, optimum, maximum : T-min -17.0 degrees C, T-opt, 19.8 degrees C, T-max 29.5 degrees C) and slow growth. Related only peripherally to Synechococcus sp. Cluster 5.2 (Marine Cluster B), the Antarctic strains represent a unique and highly adapted clade in the stable water columns of some saline Antarctic lakes.


Qin QW, Gin KY, Lee LY, Gedaria AI, Zhang S (2005) Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture. J Virol Methods 125 :49-54

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15737416

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.


Qin QW, Gin KYH, Lee LY, Gedaria AI, Zhang S (2005) Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture. Journal of Virological Methods 125 :49-54

<Go to ISI> ://000227743200007

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter (R) EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR. (c) 2004 Elsevier B.V. All rights reserved.


Rouillon G, Rivas JG, Ochoa N, Navarro E (2005) Phytoplankton composition of the stomach contents of the mussel Mytilus edulis L. from two populations : Comparison with its food supply. Journal of Shellfish Research 24 :5-14

<Go to ISI> ://000229059200002

Seasonal data on phytoplankton composition of seston and stomach contents of the mussel (Mytilus edulis Linnaeus, 1758) from two contrasting sites, an estuarine mud flat and a rocky open shore, were compared to ascertain : (a) the extent to which differential characteristics of both sites affect this composition and (b) the degree of similarity between stomach contents and microalgal composition of seston of these sites as an index reflecting the complex processes of selection taking place within the feeding-digestive system of mussels. Individuals and water samples were collected monthly from November 2001 to December 2002. when salinity, temperature, and total and organic particulate matter concentration were also recorded in the water column. Preserved samples of seston and stomach contents were analyzed by inverted microscopy according to the Utermohl method. Phytoplankton cells were counted and the different species grouped, taxonomically and, according to the habitat, into pelagic and tychopelagic. These data served to compute abundance (total cell count) and frequency index. Relative abundances of each group were compared for similarity between sampling sites and stomach and water samples in each site. Similarity analyses were performed using the index of Bray-Curtis, significant differences between samples being determined by the non parametric test of ANOSIM. Results of this test for the comparison between water and stomach contents resulted in significant differences : R = 0.68 in the estuary and R = 0.75 in the open shore area. Stomach contents presented a reduced average number of species (n = 6 in mussels from both sites) and a greater proportion of tychopelagic forms for comparison with the water samples (n = 20 and 24 in the estuary and open shore, respectively). Maximum phytoplankton density in water samples occurred in the May to October period, the group responsible for this increment being the diatoms. The stomach contents of marine mussels displayed two peaks of phytoplankton concentration in May (caused by the dinoflagellate Ensiculifera sp.) and in July (caused by the diatoms Pseudo-nitzschia pungens and Licinophora sp.). In the case of stomach contents of estuarine mussels, a single peak of abundance was recorded in the month of May and was mainly produced by Ensiculifera sp. To conclude, the main result coming from these comparisons is the increased abundance of dinoflagellates in the stomach contents relative to the corresponding seawater samples in the estuarine and open shore media. This result is discussed in the light of previous data concerning the differential utilization of species of phytoplankton by bivalve molluscs.


Rutten TP, Sandee B, Hofman AR (2005) Phytoplankton monitoring by high performance flow cytometry : a successful approach ? Cytometry A 64 :16-26

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15688354

BACKGROUND : Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage. METHODS : Phytoplankton community abundance and composition were routinely determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics. RESULTS : Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin- or phycocyanin-containing cells (cells/ml) as a function of their algal size were produced every 2 weeks on average. The phytoplankton integrated annual concentration and biomass were used as ecological indicators for overall phytoplankton status. Real-time imaging of cells in flow enabled the identification of dominant species and was applied as an early warning system for Phaeocystis spp. CONCLUSIONS : The reproducibility and count precision due to the large number of observations of the flow cytometric technique provided reliable data for monitoring long-term trends. Flow cytometrically based analyses extended the lower detection limit (<0.5 microm) of analysis beyond the capabilities of other techniques such as the relation between small and larger phytoplankton, the relation between cell counts and biomass as a function of cell size, but also the ability to monitor and report on blooms of harmful algae. A good correlation was found between concentrations (cells/ml) measured by flow cytometry and microscopy. In practice, flow cytometric analysis of a single marine sample took 15 min on average.


Rutten TPA, Sandee B, Hofman ART (2005) Phytoplankton monitoring by high performance flow cytometry : A successful approach ? Cytometry Part A 64A :16-26

<Go to ISI> ://000227272000004

Background : Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage.
Methods : Phytoplankton community abundance and composition were routinely, determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics.
Results : Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin- or Phycocyanin-containing cells (cells/ml) as a function of their algal size were produced every 2 weeks on average. The phytoplankton integrated annual concentration and biomass were used as ecological indicators for overall phytoplankton status. Real-time imaging of cells in flow enabled the identification of dominant species and was applied as an early warning system for Phaeocystis spp.
Conclusions : The reproducibility and count precision due to the large number of observations of the flow cytometric technique provided reliable data for monitoring long-term trends. Flow cytometrically based analyses extended the lower detection limit (<0.5 mum) of analysis beyond the capabilities of other techniques such as the relation between small and larger phytoplankton, the relation between cell counts and biomass as a function of cell size, but also the ability to monitor and report on blooms of harmful algae. A good correlation was found between concentrations (cells/ml) measured by flow cytometry and microscopy. In practice, flow cytometric analysis of a single marine sample took 15 min on average. (C) 2005 Wiley-Liss, Inc.


Sachidanandham R, Gin KY, Poh CL (2005) Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnol Bioeng 89 :24-31

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15540195

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states : cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.


Sachidanandham R, Gin KYH, Poh CL (2005) Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnology and Bioengineering 89 :24-31

<Go to ISI> ://000226120900004

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate HO3N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states : cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method. (C) 2004 Wiley Periodicals, Inc.


Salinas-Flores L, Paniagua-Chavez CG, Jenkins JA, Tiersch TR (2005) Cryopreservation of sperm of red abalone (Haliotis rufescens). Journal of Shellfish Research 24 :415-420

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Abalone culture, a developing industry in Baja California, Mexico, would benefit from genetic improvement and controlled breeding. The use of cryopreserved sperm would allow germplasm availability, and this study was designed to develop sperm cryopreservation protocols for red abalone Haliotis rufescens. The acute toxic effects of the cryoprotectants dimethyl sulfoxide (DMSO), propylene glycol (PG), and glycerol (GLY) were assessed after suspending sperm in different concentrations, whereby cryoprotectant treatments of 10% DMSO and 10% GLY equilibrated for 10 min yielded the highest range of motile sperm in preliminary freezing trials and were used for cryopreservation studies. To determine effective cooling rates, three freezing chambers were tested. Replicate samples of sperm from 4 males were placed in 0.5-mL French straws and frozen using a commercial freezing chamber (CFC) used for bull sperm, a programmable rate chamber (PRC), and a manually controlled styrofoam chamber (MCC). For the CFC, the cooling rate was 16 degrees C/min, from 4 degrees C to -140 degrees C. For the PRC and MCC, it was 1 degrees C/min, from -20 degrees C to -30 degrees C. The samples were held at -30 degrees C for 5 min before being plunged into liquid nitrogen (-196 degrees C) for storage, and each sample was thawed in a water bath at 45 degrees C for 8 s. The quality of thawed sperm was determined by estimating percent motility, evaluating membrane integrity using a dual-staining technique and flow cytometry, and estimating fertilization rate. Statistical analyses were performed using 2-way ANOVA where chamber and treatment were the independent variables. Sperm quality parameters were independent. For motilities, a significant interaction was noted between the cryoprotective treatment and the chamber type, whereby motilities for DMSO and GLY were higher (P = 0.0055) using MCC. Membrane integrities were significantly lower after using the PRC than the CFC or the MCC (P = 0.0167). The highest post-thaw motility (48 +/- 7%) was found using sperm suspended in 10% glycerol and frozen in the MCC. The highest percent of intact membranes (56 +/- 11%) was for sperm suspended in 10% glycerol and frozen in the CFC. The highest fertilization rate (29 +/- 10%) was with samples frozen with 10% glycerol in the CFC. The use of cryopreserved sperm from red abalone provides an alternative breeding option for culture and the protocols delineated are the first developed for this species.


Seymour JR, Patten N, Bourne DG, Mitchell JG (2005) Spatial dynamics of virus-like particles and heterotrophic bacteria within a shallow coral reef system. Marine Ecology-Progress Series 288 :1-8

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Variations in the abundance and community characteristics of virus-like particles (VLP) and heterotrophic bacteria within a shallow, near-shore coral reef were determined using flow cytometric analysis. Mean concentrations of 6.5 x 10(5) and 1.3 x 10(5) Ml(-1) were observed for VLP and bacterioplankton, respectively, although concentrations of both populations varied significantly (p < 0.05) between 4 distinct reef water types. Significant (p < 0.05) variability in the percentage of high DNA (HDNA) bacteria, applied here as an estimate of the proportion of active bacterial cells, and the virus:bacteria ratio (VBR) was also observed between different reef water types. Microscale profiles were taken in the 12 cm layer of water directly above the surface of coral colonies to determine the small-scale spatial relationships between coral colonies and planktonic microbial communities. Across these profiles, mean changes of 2- and 3.5-fold were observed for bacterioplankton and VLP communities, respectively, with VLP abundance positively correlated to bacteria in 75% of profiles. Bacterial and VLP abundance, percentage of HDNA bacteria, and VBR all generally exhibited increasing trends with proximity to the coral surface. VLP abundance was significantly higher (p < 0.05) in the 4 cm closest to the coral surface, and the VBR was higher at the coral surface than in any other zone. The patterns observed here indicate that VLP represent an abundant and dynamic community within coral reefs, are apparently coupled to the spatial dynamics of the bacterioplankton community, and may consequently significantly influence nutrient cycling rates and food-web structure within coral reef ecosystems.


Seymour JR, Seuront L, Mitchell JG (2005) Microscale and small-scale temporal dynamics of a coastal planktonic microbial community. Marine Ecology-Progress Series 300 :21-37

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The temporal dynamics of heterotrophic bacteria and Synechococcus-type cyanobacteria communities were studied in a coastal habitat characterised by strong hydrodynamic variability using 10 s (microscale) and 30 min (small-scale) sampling intervals. Flow cytometric analysis allowed for the discrimination of 3 populations of heterotrophic bacteria and the examination of the Synechococcus cell cycle. During the 11 h small-scale study, 2-fold changes in the total abundance of both the bacterial and Synechococcus communities were observed, and clear temporal patterns in the abundance, activity and cellular state of the 2 populations were evident. Cumulative sum analysis further revealed distinct periods and trends in the temporal dynamics of the bacterial and Synechococcus communities. Shifts in the abundance of all heterotrophic bacterial populations were significantly correlated to turbulent energy dissipation, No such correlation was evident for the Synechococcus population, which instead appeared to follow a diel cell cycle very similar in nature to patterns observed in other environments. In 2 microscale studies, conducted during dissimilar hydrodynamic conditions, approx. 2-fold shifts in the abundance of the bacterial and Synechococcus populations were also observed. Microscale temporal patterns were dominated by localised variability and the existence of hotspots in abundance and activity, although cumulative sum analysis also revealed more general trends, sometimes occurring over periods of several minutes. Fundamentally different patterns, in the extent of temporal variability and coupling between the different microbial populations, were observed between the microscale and small-scale studies, suggesting that intrinsically different mechanisms and responses occurred independently and simultaneously at the different temporal scales. Furthermore, the variability in microbial parameters observed over these short temporal scales indicates the profound importance of microscale and small-scale processes in the ecology of communities of marine microorganisms.


Smolarz K, Renault T, Soletchnik P, Wolowicz M (2005) Neoplasia detection in Macoma balthica from the Gulf of Gdansk : comparison of flow cytometry, histology and chromosome analysis. Diseases of Aquatic Organisms 65 :187-195

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A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B’ and C’. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B’ and C’ and on the ratio between the fluorescence value of peaks B, C, B’ and C’ in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C’. Stage 1 was characterised by a major population of cells in peak B’ and more than 10 % of cells in the C’ peak. Stage 2 consisted of a lower percentage of cells in peak B’ and more than 25 % of cells in peak C’. Stage 3 of the neoplasia was characterised by a further reduction in peak B’ and more than 40 % of cells in peak C’. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.


Smolarz K, Renault T, Soletchnik P, Wolowicz M (2005) Survey for neoplasia in Macoma balthica from the Gulf of Gdansk by flow cytometry. Diseases of Aquatic Organisms 66 :41-46

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Using flow cytometry, 234 Macoma balthica were examined during a survey to determine frequency of neoplasia in the Gulf of Gdansk (Poland). Clams were collected in 4 locations and DNA content in gill tissue cells was determined by flow cytometry using propidium iodide staining. Cell permeabilization was induced by osmotic shock. Prevalence of neoplasia ranged from 9.6 to 26.7 % depending on location. DNA content in aneuploid cells was higher than in normal dividing cells. The fluorescence value for aneuploid cells corresponded to tetraploid/pentaploid cells. Three stages of neoplasia were defined, based on the percentage of aneuploid cells determined by flow cytometry. Histopathological and cytogenetic analyses were also carried out on the same clams for comparative study. Proportions of normal and affected clams detected using flow cytometry were similar to those identified using both methods. In the present study, no clear relationship was demonstrated between prevalence of neoplasia and pollutant detection in the different sampling sites.


Snyder RV, Guerrero MA, Sinigalliano CD, Winshell J, Perez R, Lopez JV, Rein KS (2005) Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis. Phytochemistry 66 :1767-1780

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16051286

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.


Sobrino C, Montero I, Lubian LM (2005) Effect of UV-A and UV-B on diel patterns of growth and metabolic activity in Nannochloris atomus cultures assessed by flow cytometry. Marine Ecology-Progress Series 293 :29-35

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The aim of this study was to assess the effect of UV-A (320 to 400 nm) and UV-B (280 to 320 nm) radiation on diel patterns of growth and metabolic activity of the marine picoplankter Nannochloris atomus using flow cytometry. N, atomus cells exposed to PAR (400 to 700 nm), PAR+UV-A and PAR+UV-A+UV-B showed clear diel patterns in cell size, chlorophyll fluorescence and metabolic activity, the latter being measured by a fluorescein diacetate-based cell esterase activity assay. For all spectral treatments, patterns increased during the day and decreased during the night, with minima near dawn and maxima near dusk. In addition, cell division was tightly phased to the light dark (L:D) cycle, occurring soon after dark. Exposure to UVR did not alter the synchrony of the parameters measured, but the extent of variation between dawn and dusk was dependent on the spectral conditions. Chlorophyll autofluorescence and metabolic activity decreased to a larger extent when cells were exposed to UV-B than in treatments where UV-B was excluded. In contrast, the cell size was larger under the treatment including UV-A+UV-B than under the treatment including only UV-A. These results show that UV-B damage can decrease growth and metabolic activity in N. atomus without altering the synchronization of the diel patterns, and contribute to a better understanding of phytoplankton behavior under UVR exposures.


Sommaruga R, Hofer JS, Alonso-Saez L, Gasol JM (2005) Differential sunlight sensitivity of picophytoplankton from surface Mediterranean Coastal Waters. Appl Environ Microbiol 71 :2154-2157

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15812050

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.


Stoffel CL, Kathy RF, Rowlen KL (2005) Design and characterization of a compact dual channel virus counter. Cytometry Part A 65A :140-147

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Background : Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard" Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer.
Methods : A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels.
Results : The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay.
Conclusions : The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (similar to 100 mu l). (c) 2005 Wiley-Liss, Inc.


Stoffel CL, Rowlen KL (2005) Data analysis for a dual-channel virus counter. Analytical Chemistry 77 :2243-2246

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A simple algorithm is presented for quantitative analysis of simultaneous events on a dual-channel flow cytometer designed specifically for virus counting. The algorithm, which is based on matrix analysis of burst lag times, was evaluated using baculovirus samples that had previously been quantified by the plaque titer method. The results indicated statistical reliability for the algorithm, with three of six samples yielding the same value, within error, for viruses per unit volume as the plaque titer. The other three samples yielded values within a factor similar to 2, which was deemed acceptable given the limitations of the plaque titer method.


Tadonleke RD, Planas D, Lucotte A (2005) Microbial food webs in boreal humic lakes and reservoirs : Ciliates as a major factor related to the dynamics of the most active bacteria. Microbial Ecology 49 :325-341

<Go to ISI> ://000230310200016

In order to assess the factors that determine the dynamics of bacteria with high nucleic acid content in aquatic systems, we (i) conducted 24-h in situ dialysis experiments, involving different fractions of plankton and unfiltered water and (ii) examined empirical relationships between bacteria and both abiotic factors and protists, in boreal humic freshwaters (reservoir and lakes) in the James Bay region (Quebec, Canada). Bacteria were subdivided into two subgroups on the basis of their nucleic acid content assessed by flow cytometry. The abundance of bacteria with the highest nucleic acid content and high light scatter (HNA-hs) was significantly correlated, across sites, to bacterial production, whereas bacteria with lower nucleic acid content (LNA) and total bacteria were not. In addition, HNA-hs growth was higher and more variable than LNA growth, indicating that HNA-hs were the most dynamic bacteria. Heterotrophic nanoflagellate and ciliate biomass represented, on average, 5 and 13% of bacterial biomass, respectively. Both in ambient waters and in experiments, ciliates were significantly and negatively correlated with bacteria, whereas heterotrophic nanoflagellates, likely under the grazing pressure from ciliates and metazooplankton, were not. Among ciliates, Cyclidium glaucoma appeared to play an important role. Its growth was significantly and negatively correlated to that of HNA-hs but not to that of LNA. In ambient waters, the abundance of this species explained 56% of the variations in HNA-hs abundance and only 27% of those for LNA. The abundances of total bacteria and LNA significantly increased with chlorophyll a, whereas those of HNA-hs did not. In addition, during the experiments, the estimated potential losses of HNAhs significantly increased with the initial abundance of C. glaucoma. These results suggest selective removal of the most dynamic bacteria by C glaucoma and indicate that ciliates may play an important role in the dynamics of active bacteria in natural waters. These findings suggest the existence, within the aquatic microbial food webs, of keystone species that are very important in regulating the activity structure of bacteria.


Tadonleke RD, Planas D, Lucotte M (2005) Microbial food webs in boreal humic lakes and reservoirs : ciliates as a major factor related to the dynamics of the most active bacteria. Microb Ecol 49 :325-341

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15965722

In order to assess the factors that determine the dynamics of bacteria with high nucleic acid content in aquatic systems, we (i) conducted 24-h in situ dialysis experiments, involving different fractions of plankton and unfiltered water and (ii) examined empirical relationships between bacteria and both abiotic factors and protists, in boreal humic freshwaters (reservoir and lakes) in the James Bay region (Quebec, Canada). Bacteria were subdivided into two subgroups on the basis of their nucleic acid content assessed by flow cytometry. The abundance of bacteria with the highest nucleic acid content and high light scatter (HNA-hs) was significantly correlated, across sites, to bacterial production, whereas bacteria with lower nucleic acid content (LNA) and total bacteria were not. In addition, HNA-hs growth was higher and more variable than LNA growth, indicating that HNA-hs were the most dynamic bacteria. Heterotrophic nanoflagellate and ciliate biomass represented, on average, 5 and 13% of bacterial biomass, respectively. Both in ambient waters and in experiments, ciliates were significantly and negatively correlated with bacteria, whereas heterotrophic nanoflagellates, likely under the grazing pressure from ciliates and metazooplankton, were not. Among ciliates, Cyclidium glaucoma appeared to play an important role. Its growth was significantly and negatively correlated to that of HNA-hs but not to that of LNA. In ambient waters, the abundance of this species explained 56% of the variations in HNA-hs abundance and only 27% of those for LNA. The abundances of total bacteria and LNA significantly increased with chlorophyll a, whereas those of HNA-hs did not. In addition, during the experiments, the estimated potential losses of HNA-hs significantly increased with the initial abundance of C. glaucoma. These results suggest selective removal of the most dynamic bacteria by C. glaucoma and indicate that ciliates may play an important role in the dynamics of active bacteria in natural waters. These findings suggest the existence, within the aquatic microbial food webs, of keystone species that are very important in regulating the activity structure of bacteria.


Takahashi H, Nagai T, Goto A (2005) Hybrid male sterility between the fresh- and brackish-water types of ninespine stickleback Pungitius pungitius (Pisces, Gasterosteidae). Zoological Science 22 :35-40

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Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback coexist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F, hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as ’gametic sterility,’ with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).


Toepel J, Langner U, Wilhelm C (2005) Combination of flow cytometry and single cell absorption spectroscopy to study the phytoplankton structure and to calculate the chl a specific absorption coefficients at the taxon level. Journal of Phycology 41 :1099-1109

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The phytoplankton community structure of a hypertrophic lake was quantitatively determined with the aid of flow cytometry. The flow cytometry signals were calibrated to obtain cell-specific information, such as the chl a content and the biovolume per cell. The reliability of this method was tested with laboratory cultures. The results of the phytoplankton structure in a hypertrophic lake with respect to chl distribution in the different algal groups obtained by flow cytometry were compared with the results from HPLC pigment fingerprinting. Both methods yield the percentage contribution of the different algal groups to total chl a. The chl a specific absorption coefficient of the phytoplankton (a(Phy)(*)) was determined via visible (VIS) spectroscopy of samples taken from a hypertrophic lake (Auensee) in 2003. The results indicated that a(Phy)(*) of the total cell suspension is dependent on the phytoplankton structure as well as on environmental factors. The linear relationship between a(Phy)(*) at 675 nm and the product of the chl a content per cell and the biovolume offered the possibility to normalize phytoplankton absorption spectra to acquire the taxon-specific a(Phy)(*). The estimated a(Phy)(*) (675 nm) values were used to normalize single cell absorption spectra at this wavelength to obtain the a(Phy)(*) between 400 and 750 nm for representatives of the major algal groups. Our measurements show that the absorption coefficient for the whole phytoplankton community varies within the season. Finally, we used the a(Phy)(*) and the chl a distribution to calculate the light absorption of each algal group in the hypertrophic lake.


Touron A, Berthe T, Pawlak B, Petit F (2005) Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay. Research in Microbiology 156 :541-553

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From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary (France). The 3 serovars most frequently found were S. enterica serovar Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella in estuarine water and sediment samples. The target gene used was the phase 1 flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4 primers was first used to amplify an 890-bp sequence of the fliC gene, and then a second set of 3 primers was used for the nested PCR. The nmPCR method has been successfully tested for 2:3 serovars, 13 of which are of epidemiological significance. The detection limit of the assay, without any pre-enrichment step, was estimated at 1 CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on estuarine water seeded with a Salmonella strain. When the nmPCR was used together with the classical culture method in environmental samples, it gave additional positive results for 11.3% of the sediment samples and 20% of the water samples despite a high background of other bacteria. Overall, the results demonstrated that this molecular approach informed us about the contamination by Salmonella of estuarine water and sediment samples. Positive amplifications suggested the presence of Salmonella DNA and could thus provide information about a recent (culturable) or past (non-culturable, released DNA) contamination of environmental samples by this pathogenic bacteria. (c) 2005 Elsevier SAS. All rights reserved.


Troussellier M, Got P, Mboup M, Corbin D, Giuliano L, Cappello S, Bouvy M (2005) Daily bacterioplankton dynamics in a sub-Saharan estuary (Senegal River, West Africa) : a mesocosm study. Aquatic Microbial Ecology 40 :13-24

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Temporal variability in bacterial communities was studied on a daily scale in the estuarine part of the largest river on the West African coast, the Senegal River. Duplicate mesocosms (3 m(3) volume) were placed in the upper part of the estuary at the end of the dry season (May 2002) and treated with low and high inorganic nutrient (N and P) enrichment. High nutrient additions were followed by a 7-fold increase in phytoplankton biomass and a 6-fold increase in bacterial abundance after 4 and 9 d, respectively. Heterotrophic nanoflagellates (HNF) showed their maximal abundance (1 x 10(6) ml(-1)) 2 d after the bacterial peak. The low bacteria to flagellate ratios recorded on Day 10 may suggest enhanced bacterivory from HNF. Simultaneous measurements of growth and grazing rates on bacteria during the bacterial growth phase were performed with the dilution method and seemed to indicate that the HNF community was capable of quickly controlling bacterial development. However, estimates of the carbon demand of HNF during their growth phase (915 mu g C l(-1) d(-1)) appeared to be more elevated than the bacterial carbon production (63% of the HNF carbon demand). To cover HNF carbon requirements, an alternative/complementary prey might be the picophytoplanktonic cells, which were very numerous during the study (i.e. 85 % of the total phytoplankton count). During the period of high grazing pressure, bacterial populations were characterized by higher specific activity (from tritiated thymidine incorporation) and culturability (from plate counts). The presence of very large bacteria, as detected by epifluorescence microscopy and flow cytometry measurements, may be an escape response from flagellate grazing. 16S rDNA sequences from bacterial isolates showed the presence of 2 types of taxonomic units (Vibrio natriegens- and Flexibacter maritimus-like bacteria), which can be considered by their forms and growth rates to be strains that have developed strategies to protect against grazing. Thus, as demonstrated in various temperate systems, predation by HNF in estuarine tropical ecosystems may also be of importance in shaping the structure and functions of the bacterial community.


Uchiyama T, Abe T, Ikemura T, Watanabe K (2005) Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23 :88-93

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15608629

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.


van de Poll WH, van Leeuwe MA, Roggeveld J, Buma AGJ (2005) Nutrient limitation and high irradiance acclimation reduce PAR and UV-induced viability loss in the Antarctic diatom Chaetoceros brevis (Bacillariophyceae). Journal of Phycology 41 :840-850

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The effects of high PAR (400-700 nm), UVA (315-400 nm), and UVB (280-315 nm) radiation on viability and photosynthesis were investigated for Chaetoceros brevis Schutt. This Antarctic marine diatom was cultivated under low, medium, and high irradiance and nitrate, phosphate, silicate, and iron limitation before exposure to a simulated surface irradiance (SSI) treatment, with and without UVB radiation. Light-harvesting and protective pigment composition and PSII parameters were determined before SSI exposure, whereas viability was measured by flow cytometry in combination with a viability stain after the treatment. Recovery of PSII efficiency was measured after 20 h in dim light in a separate experiment. In addition, low and high irradiance acclimated cells were exposed outdoors for 4 h to assess the effects of natural PAR, UVA, and UVB on viability. Low irradiance acclimated cells were particularly sensitive to photo induced viability loss, whereas no viability loss was found after acclimation to high irradiance. Furthermore, nutrient limitation reduced sensitivity to photo induced viability loss, relative to nutrient replete conditions. No additional viability loss was found after UVB exposure. Sunlight exposed cells showed no additional UVB effect on viability, whereas UVA and PAR significantly reduced the viability of low irradiance acclimated cells. Recovery of PSII function was nearly complete in cultures that survived the light treatments. Increased resistance to high irradiance coincided with an increased ratio between protective- and light-harvesting pigments before the SSI treatment, demonstrating the importance of nonphotochemical quenching by diatoxanthin for survival of near-surface irradiance. We conclude that a sudden transfer to high irradiance can be fatal for low irradiance acclimated C. brevis.


van Oijen T, Veldhuis MJW, Gorbunov MY, Nishioka J, van Leeuwe MA, de Baara HJW (2005) Enhanced carbohydrate production by Southern Ocean phytoplankton in response to in situ iron fertilization. Marine Chemistry 93 :33-52

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Storage carbohydrates (e.g., water-extractable beta-1,3-D-glucan in diatoms) are of key importance for phytoplankton growth in a variable light climate, because they facilitate continued growth of the cells in darkness by providing energy and carbon skeletons for protein synthesis. Here, we tested the hypothesis that synthesis of storage carbohydrates by phytoplankton in the Southern Ocean is reduced by low iron and light availability. During the EisenEx/CARbon dioxide Uptake by the Southern Ocean (CARUSO) in situ iron enrichment experiment in the Atlantic sector of the Southern Ocean in November 2000, we studied the dynamics of water-extractable carbohydrates in the particulate fraction over the period of 3 weeks following the iron release. The areal amount (integral between 0- and 100-m depth) of carbohydrates increased from 1400 to 2300 mg m(-2) inside the iron-enriched patch, while remaining roughly constant in the surrounding waters. Most of the increase inside the patch was associated with the fraction of large (>10 mum) phytoplankton cells, consistent with the shift in the community structure towards larger diatoms. Deck incubations at 60% of the ambient irradiance revealed that the diurnal chlorophyll a (Chl a)-specific production rates of water-extractable polysaccharides were significantly higher for "in-patch" than for "out-patch" samples (0.5 vs. 0.3 mug C [mug Chl a](-1) h(-1), respectively). Together with the higher photochemical efficiency of photosystent II (F-v/F-m), this indicates enhanced photosynthetic performance in response to iron fertilization. In addition, the nocturnal polysaccharide consumption rates were also enhanced by iron release, causing a striking increase in the diel dynamics of polysaccharide concentration. An iron-stimulated increase in diel dynamics was also observed in the fluorescence and size of pico- and nanophytoplankton cells (measured by flow cytometry) and is indicative of enhanced phytoplankton growth. Diurnal polysaccharide production by phytoplankton inside the patch was light-limited when they were incubated at intensities below ca. 200 mumol m(-2) s(-1) (daytime average). These irradiance levels correspond to those at 20- to 30-m depth in situ, whereas the upper mixed layer was frequently several-fold deeper due to storms. Therefore, these first measurements of phytoplankton carbohydrates during an in situ iron release experiment have revealed that both light and iron availability are the key factors controlling the synthesis of storage carbohydrates in phytoplankton and, hence, the development of diatom blooms in the Southern Ocean. (C) 2004 Elsevier B.V. All rights reserved.


Vazquez-Dominguez E, Casamayor EO, Catala P, Lebaron P (2005) Different marine heterotrophic nanoflagellates affect differentially the composition of enriched bacterial communities. Microbial Ecology 49 :474-485

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We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates : Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1) ; after that the FL1 decreased when bacteria attained maximal concentrations ; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.


Veldhuis MJW, Brussaard CPD, Noordeloos AAM (2005) Living in a Phaeocystis colony : a way to be a successful algal species. Harmful Algae 4 :841-858

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Evidence is provided showing that in two species of Phaeocystis (P. globosa and P pouchetii) the colonial cells possess a much higher growth rate than the single cells when grown under identical conditions. Based on the DNA-cell-cycle method gross growth rate of colony cells exceeded those of co-occurring single cells by a factor 1.5 up to 3.8. The dominance of colonies in blooms of Phaeocystis can therefore be primarily due to their significantly high growth rate allowing a rapid bloom formation.
Both Phaeocystis species showed ultradian growth but differed in timing of the initiation of the second DNA replication phase. In both species the first DNA-replication period started at the end of the (local) light period and was completed in the early dark period. In P globosa this was immediately followed by the second DNA-replication period (first half of the dark period). In P. pouchetii this process was delayed by ca. 12 h until the middle of the light period (local noon).
Flow cytometric analysis of the cell size and chlorophyll fluorescence showed little variation in colony and single cells of P. pouchetii. In contrast, colonies of P. globosa showed often the presence of two cell morphs, co-occurring in the same colony. The size of both morphs was identical but they differed in chlorophyll fluorescence up to a factor 4. In general the high chlorophyll cell morph dominated (>70% of the total colony cells). Both colony cell morphs were observed in cultures, mesocosms differing in N/P ratio but also in the field. (C) 2005 Elsevier B.V. All rights reserved.


Veldhuis MJW, Timmermans KR, Croot P, van der Wagt B (2005) Picophytoplankton ; a comparative study of their biochemical composition and photosynthetic properties. Journal of Sea Research 53 :7-24

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Picophytoplankton are a small or major component of the phytoplankton community and present in all oceanic systems, from pole to pole. They dominate in the low chlorophyll biomass areas, such as the (sub)tropical regions, but also contribute considerably (up to 20%) in the high chlorophyll biomass areas. The ecosystems of occurrence contrast significantly in physical and chemical settings. This includes a strongly mixed upper water column replete in nutrients as well as a strongly thermally stratified euphotic zone depleted in nutrients at the surface with a steep inverse light and nutrient gradient. These changes impose a strong impact on the composition of the picophytoplankton community but also on the biochemical and physiological properties of the species present. In particular, the pigmentation and cellular carbon, nitrogen and phosphorus quota and requirement will differ from a stratified compared to a well-mixed water column. As a result no characteristic values for the parameters required for this specific algal group in a global phytoplankton carbon model (the SWAMCO model, Lancelot et al. (2000), Deep-Sea Res. 1, 47, 162 1) can be given. In the present paper an inventory is made of the biochemical, physiological and photosynthetic parameters of two species of cyanobacteria (Prochlorococcus and Synechococcus) and the pico-size class fraction of the eukaryote phytoplankton component. Other groups of phytoplankton, such as diatoms, Trichodesmium, Phaeocystis and coccolithophorids, will be discussed in separate papers in this issue. This inventory is a mixture of laboratory experiments using well-defined algal populations as well as data derived from field surveys including a mixture of species. Where possible, the relevance of the parameters will be discussed in relation to the nature of the physico-chemical conditions of the area of occurrence. (C) 2004 Elsevier B.V. All rights reserved.


Wang X, Chan RK, Cheng AS (2005) Underwater cytometer for in situ measurement of marine phytoplankton by a technique combining laser-induced fluorescence and laser Doppler velocimetry. Opt Lett 30 :1087-1089

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We present a new type of flow cytometer that can operate underwater for a long time, as long as days, for measuring the size distribution, concentration, and biomass of marine phytoplankton. The major improvement of the instrument over existing techniques is the elimination of sample preparation, which is achieved with a laser Doppler crossed-beam arrangement for both defining a measurement volume and measuring the speed of the particle traversing it. By simultaneously sampling the laser-induced fluorescence signal and the Doppler signals, the technique can discriminate sizes of phytoplankton.


Welch TJ, Wiens GD (2005) Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge. Dis Aquat Organ 67 :267-272

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16408843

A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.


Xu DH, Klesius PH, Shoemaker CA (2005) Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts. J Fish Dis 28 :213-220

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This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.


Zhu F, Massana R, Not F, Marie D, Vaulot D (2005) Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. Fems Microbiology Ecology 52 :79-92

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A quantitative PCR (QPCR) assay based on the use of SYBR Green I was developed to assess the abundance of specific groups of picoeukaryotes in marine waters. Six primer sets were designed targeting four different taxonomic levels : domain (Eukaryota), division (Chlorophyta), order (Mamiellales) and genus (Bathycoccus, Micromonas, and Ostreococcus). Reaction conditions were optimized for each primer set which was validated in silico, on agarose gels, and by QPCR against a variety of target and non-target cultures. The approach was tested by estimating gene copy numbers for Micromonas, Bathycoccus, and Ostreococcus in seawater samples to which cultured cells were added in various concentrations. QPCR was then used to determine that rRNA gene (rDNA) copy number varied from one to more than 12,000 in 18 strains of phytoplankton. Finally, QPCR was applied to environmental samples from a Mediterranean Sea coastal site and the results were compared to those obtained by Fluorescent in situ hybridization (FISH). The data obtained demonstrate that Chlorophyta and more specifically Mamiellales were important in these waters, especially during the winter picoplankton bloom. The timing of major abundance peaks of the targeted species was similar by QPCR and FISH. When used in conjunction with other techniques such as FISH or gene clone libraries, QPCR appears as very promising to quickly obtain data on the ecological distribution of important phytoplankton groups. Data interpretation must take into account primer specificity and the varying rRNA gene copy number among eukaryotes. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.


Zubkov MV, Tarran GA (2005) Amino acid uptake of Prochlorococcus spp. in surface waters across the South Atlantic Subtropical Front. Aquatic Microbial Ecology 40 :241-249

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To test the hypothesis that surface-living Prochlorococcus spp. (Pro) cyanobacteria metabolism declines towards the boundaries of its natural habitat, a latitudinal transect of surface waters was sampled from the centre of the Southern Atlantic Gyre (SAG, 20 to 35 degrees S) to the South Subtropical Frontal Zone (SSFZ, 35 to 45 degrees S). Along this transect, amino acid uptake rates of Pro, Synechococcus spp. (Syn) and an average bacterioplankton cell were determined using S-35-methionine precursor and flow-cytometry sorting, with methionine uptake rate as an index of cellular metabolic activity. Methionine and possibly other amino acids were a very minor nutrient source for Syn, while their contribution to Pro production was significant. Contrary to expectations, the mean methionine uptake rate per Pro cell in the SSFZ was about 3 times higher than in the SAG. The uptake rates per unit Pro biomass were equal to or higher than that of an average bacterioplankton cell in both the SAG and SSFZ. About 20 and 5% of total bacterioplankton consumption of amino acids could be assigned to Pro in the SAG and SSFZ, respectively. Methionine and leucine turnover rates were 3.5 and 3 times higher in the SSFZ than in the SAG, respectively. These results suggest that Pro remained highly metabolically active and acquired more methionine at its habitat boundaries, despite higher rates of bacterioplankton activity and therefore greater competition, as well as exposure to deep water mixing, low light and low temperature conditions.