mardi 21 avril 2009
par   G. Grégori

Almeida WI, Vieira RP, Cardoso AM, Silveira CB, Costa RG, Gonzalez AM, Paranhos R, Medeiros JA, Freitas FA, Albano RM, Martins OB (2009) Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay. Extremophiles 13 :263-271

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Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd-Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, alpha-Proteobacteria (13.4%), beta-Proteobacteria (16.3%), gamma-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.

Arzul I, Gagnaire B, Bond C, Chollet B, Morga B, Ferrand S, Robert M, Renault T (2009) Effects of temperature and salinity on the survival of Bonamia ostreae, a parasite infecting flat oysters Ostrea edulis. Dis Aquat Organ 85 :67-75


Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting the flat oyster Ostrea edulis. The parasite has been described in various ecosystems from estuaries to open sea, but no clear correlation has yet been demonstrated between disease development and environmental parameters. In this study, the effect of temperature and salinity on the survival of purified parasites maintained in vitro in seawater was investigated by flow cytometry. Purified parasites were incubated in various seawater media (artificial seawater, natural seawater, seabed borewater) at various temperatures (4, 15 and 25 degrees C) and subjected to a range of salinities from 5 to 45 g l(-1). Parasites were collected after 12, 24 and 48 h of incubation for flow cytometry analyses including estimation of parasite mortality and parasite viability through detection of non-specific esterase activities. Artificial seawater appeared unsuitable for parasite survival, and results for all media showed a significantly lower survival at 25 degrees C compared to 4 degrees C and 15 degrees C. Moreover, high salinities (> or = 35 g l(-1)) favoured parasite survival and detection of esterase activities. Flow cytometry appears to be a suitable technique to investigate survival and activities of unicellular parasites like B. ostreae under varied conditions. Although these results contribute to a better understanding of existing interactions between the parasite B. ostreae and its environment, validation through epidemiological surveys in the field is also needed.

Auguet JC, Montanie H, Hartmann H, Lebaron P, Casamayor EO, Catala P, Delmas D (2009) Potential Effect of Freshwater Virus on the Structure and Activity of Bacterial Communities in the Marennes-Ol,ron Bay (France). Microbial Ecology 57 :295-306

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Batch culture experiments using viral enrichment were conducted to test the response of a coastal bacterial community to autochthonous (i.e., co-existing) or allochthonous riverine viruses. The effects of viral infections on bacterial dynamics and activity were assessed by epifluorescence microscopy and thymidine incorporation, respectively, whereas the effect of viral infection on bacterial community composition was examined by polymerase chain reaction-single strand conformation polymorphism 16S ribosomal RNA fingerprinting. The percentages of high nucleic acid-containing cells, evaluated by flow cytometry, were significantly correlated (r (2) = 0.91, n = 12, p < 0.0001) to bacterial production, making this value a good predictor of active cell dynamics along the study. While confinement and temperature were the two principal experimental factors affecting bacterial community composition and dynamics, respectively, additions of freshwater viruses had significant effects on coastal bacterial communities. Thus, foreign viruses significantly reduced net bacterial population increase as compared to the enrichment treated with inactivated virus. Moreover, freshwater viruses recurrently and specifically affected bacterial community composition, as compared to addition of autochthonous viruses. In most cases, the combined treatment viruses and freshwater dissolved organic matter helped to maintain or even enhance species richness in coastal bacterial communities in agreement to the ’killing the winner’ hypothesis. Thus, riverine virus input could potentially influence bacterial community composition of the coastal bay albeit with modest modification of bulk bacterial growth.

Barer MR, Kaprelyants AS, Weichart DH, Harwood CR, Kell DB (1998) Microbial stress and culturability : conceptual and operational domains. Microbiology-Uk 144 :2009-2010

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Beineke A, Siebert U, Wohlsein P, Baumgartner W (2009) Immunology of whales and dolphins. Vet Immunol Immunopathol

The increasing disease susceptibility in different whale and dolphin populations has led to speculation about a possible negative influence of environmental contaminants on the immune system and therefore on the health status of marine mammals. Despite current efforts in the immunology of marine mammals several aspects of immune functions in aquatic mammals remain unknown. However, assays for evaluating cellular immune responses, such as lymphocyte proliferation, respiratory burst as well as phagocytic and cytotoxic activity of leukocytes and humoral immune responses have been established for different cetacean species. Additionally, immunological and molecular techniques enable the detection and quantification of pro- and anti-inflammatory cytokines in lymphoid cells during inflammation or immune responses, respectively. Different T and B cell subsets as well as antigen-presenting cells can be detected by flow cytometry and immunohistochemistry. Despite great homologies between marine and terrestrial mammal lymphoid organs, some unique anatomical structures, particularly the complex lymphoepithelial laryngeal glands in cetaceans represent an adaptation to the marine environment. Additionally, physiological changes, such as age-related thymic atrophy and cystic degeneration of the "anal tonsil" of whales have to be taken into account when investigating these lymphoid structures. Systemic morbillivirus infections lead to fatalities in cetaceans associated with generalized lymphoid depletion. Similarly, chronic diseases and starvation are associated with a loss of functional lymphoid cells and decreased resistance against opportunistic infections. There is growing evidence for an immunotoxic effect of different environmental contaminants in whales and dolphins, as demonstrated in field studies. Furthermore, immunomodulatory properties of different persistent xenobiotics have been confirmed in cetacean lymphoid cells in vitro as well as in animal models in vivo. However, species-specific differences of the immune system and detoxification of xenobiotics between cetaceans and laboratory rodents have to be considered when interpreting these toxicological data for risk assessment in whales and dolphins.

Bosshard F, Berney M, Scheifele M, Weilenmann HU, Egli T (2009) Solar disinfection (SODIS) and subsequent dark storage of Salmonella typhimurium and Shigella flexneri monitored by flow cytometry. Microbiology 155 :1310-1317


Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation : apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m(-2)) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (<400 W m(-2)).

Brussaard CP (2009) Enumeration of bacteriophages using flow cytometry. Methods Mol Biol 501 :97-111


Rapid identification and enumeration of the numerically important bacteriophages has been till recently a major limitation for studies of virus ecology. The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has changed this. The flow cytometric method allows the detection and discrimination of a wide variety of viruses of different morphology, genome type, and size. The present paper describes an optimized protocol for the enumeration of bacteriophages using a standard benchtop flow cytometer.

Buma AGJ, Sjollema SB, van de Poll WH, Klamer HJC, Bakker JF (2009) Impact of the antifouling agent Irgarol 1051 on marine phytoplankton species. Journal of Sea Research 61 :133-139

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In the present study we tested the hypothesis that environmental concentrations of the antifouling agent Irgarol 1051, as measured in coastal Western European waters, affect marine phytoplankton performance. The impact of Irgarol was investigated in the phytoplankton species Thalassiosira weissflogii, Emiliania huxleyi, Tetraselmis sp. and Fibrocapsa japonica. EC50 concentrations for growth, effective quantum yield of PSII and viability were calculated from dose response relationships established during 72 h exposures to six Irgarol concentrations. Furthermore, the biological recuperation from a temporary exposure to a high Irgarol concentration (39.47 nM l(-1)) was monitored. Growth rates and effective quantum yield were strongly affected by Irgarol, however viability loss was never observed. EC50 values differed five fold between species and ranged from 0.43 to 2.38 nM for effective quantum yield and from 0.46 to 2.44 nM for growth rate. For all species, complete biological recuperation was shown within 3-4 days after the Irgarol treatment, both for effective quantum yield and growth rate. All calculated EC50 values and EC20 Values fall within the Irgarol concentration range measured in Western European coastal waters. We therefore conclude that present day Irgarol 1051 levels may affect the in situ performance of marine phytoplankton in this area. (C) 2008 Published by Elsevier B.V.

Caro A, Got P, Bouvy M, Troussellier M, Gros O (2009) Long term starvation of a host bivalve (Codakia orbicularis, Lucinidae) and effects on its symbiont population. Appl Environ Microbiol 75 :3304–3313


The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill-endosymbionts, were starved (0.22microm filtered artificial seawater) for a long term experiment (4 months). The effects of starvation were observed using TEM, CARD-FISH and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with the loss of one third of the bacterial population each month as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content while most of the symbionts maintained a high respiratory activity (CTC method). Progressively, the number of symbiont subpopulations was reduced and the subsequent multigenomic state characteristic of this symbiont in freshly collected clams turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes while large symbionts disappeared and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival in these starvation conditions was linked to symbiont digestion as the main nutritional source.

Cunliffe M, Salter M, Mann PJ, Whiteley AS, Upstill-Goddard RC, Murrell JC (2009) Dissolved organic carbon and bacterial populations in the gelatinous surface microlayer of a Norwegian fjord mesocosm. FEMS Microbiol Lett 299 :248-254


The sea surface microlayer is the interfacial boundary layer between the marine environment and the troposphere. Surface microlayer samples were collected during a fjord mesocosm experiment to study microbial assemblage dynamics within the surface microlayer during a phytoplankton bloom. Transparent exopolymer particles were significantly enriched in the microlayer samples, supporting the concept of a gelatinous surface film. Dissolved organic carbon and bacterial cell numbers (determined by flow cytometry) were weakly enriched in the microlayer samples. However, the numbers of Bacteria 16S rRNA genes (determined by quantitative real-time PCR) were more variable, probably due to variable numbers of bacterial cells attached to particles. The enrichment of transparent exopolymer particles in the microlayer and the subsequent production of a gelatinous biofilm have implications on air-sea gas transfer and the partitioning of organic carbon in surface waters.

Cunliffe M, Whiteley AS, Newbold L, Oliver A, Schafer H, Murrell JC (2009) Comparison of bacterioneuston and bacterioplankton dynamics during a phytoplankton bloom in a fjord mesocosm. Appl Environ Microbiol 75 :7173-7181


The bacterioneuston is the community of Bacteria present in surface microlayers, the thin surface film that forms the interface between aquatic environments and the atmosphere. In this study we compared bacterial cell abundances and bacterial community structures of the bacterioneuston and the bacterioplankton (from the subsurface water column) during a phytoplankton bloom mesocosm experiment. Bacterial cell abundance, determined by flow cytometry, followed a typical bacterioplankton response to a phytoplankton bloom, with Synechococcus and high-nucleic acid content (HNA) bacterial cell numbers initially falling, probably due to selective protist grazing. Subsequently HNA and low-nucleic acid content bacterial cells increased in abundance, but Synechococcus cells did not. There was no significant difference between bacterioneuston and bacterioplankton cell abundances during the experiment. Conversely, distinct and consistent differences between the bacterioneuston and the bacterioplankton community structures were observed. This was monitored simultaneously by Bacteria 16S rRNA gene terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis. The conserved patterns of community structure observed in all of the mesocosms indicate that the bacterioneuston is distinctive and nonrandom.

Du L, Zhu T, Li L, Cai S, Zhao B, Gu Q (2009) Cytotoxic sorbicillinoids and bisorbicillinoids from a marine-derived fungus Trichoderma sp. Chem Pharm Bull (Tokyo) 57 :220-223


Four sorbicillinoids (1-4) and seven bisorbicillinoids (5-11), including two new compounds, 6-demethylsorbicillin (1) and 10,11-dihydrobisvertinolone (6), were isolated from a marine-derived fungus Trichoderma sp. Their cytotoxic activities against HL-60 cell line were evaluated by Sulforhodamine B (SRB) assay method and flow cytometric analysis.

Duhamel S, Gregori G, Van Wambeke F, Nedoma J (2009) Detection of extracellular phosphatase activity at the single-cell level by enzyme-labeled fluorescence and flow cytometry : the importance of time kinetics in ELFA labeling. Cytometry A 75 :163-168


ELF97 phosphate (ELF-P) is a useful compound for assessing the phosphorus-related status of planktonic aquatic populations. The technique has been successfully applied to phytoplankton and more recently to heterotrophic prokaryotes in both freshwater and marine samples. We have used a recently developed protocol that enables the detection by flow cytometry of ELF alcohol (ELFA), the product of ELF-P hydrolysis. This protocol allows for identification of the fraction of cells able to express phosphatase activity (i.e., ELFA-labeled). This protocol is also very valuable in the study of time kinetics in this ELFA-labeling. The percentage of ELFA-labeled cells, the relative median ELFA fluorescence per cell, and the absolute ELFA fluorescence were determined in both freshwater (lake) and marine samples. The incubation time necessary to reach a stable percentage of active cells with maximal fluorescence intensity varied widely among samples. We highlight very subtle but important problems of discrimination between active and nonactive cells and of estimation of per-cell activity and we underline the importance of studying time kinetics of ELFA-labeling to determine the appropriate incubation time and thus making sample comparisons more relevant. Working on time kinetics of ELFA-labeling is promising for phosphomonoester hydrolysis rate determination at single cell level.

Favret KP, Lynn JW (2009) Flow-Cytometric Analyses of Viability Biomarkers in Pesticide-Exposed Sperm of Three Aquatic Invertebrates. Arch Environ Contam Toxicol

Toxicity studies on sperm often use fertilization success as the end point. This type of assay can be affected by sperm density, egg quality, and sperm-egg compatibility. Testing sperm viability biomarkers with flow cytometry is a fast, high-throughput technique for seminal analysis. In this study, we detected sperm viability biomarkers with several fluorescent reporter dyes using flow cytometry in three aquatic invertebrates (Crassostrea virginica, Dreissena polymorpha, and Lytechinus variegatus) after exposure to a pesticide and herbicide. The pesticide, Bayluscide, appeared to affect mitochondrial membrane potential in the sperm of all three species, as measured with MitoTracker Red CMXRos((R)). A decrease in the percentage of sperm stained with SYBR((R))-14 (indicating uncompromised plasma membrane) was observed in C. virginica and D. polymorpha sperm exposed to Bayluscide, but propidium iodide staining (indicating compromised plasma membranes) appeared to be inhibited by Bayluscide. Acrosome-reacted sperm, as measured by FITC-PNA, decreased after Bayluscide exposure in C. virginica and D. polymorpha sperm. The herbicide, Roundup Ready To-Use-Plus((R)), did not affect the overall percentages of sperm stained with MitoTracker but did cause an increase in MitoTracker fluorescence intensity at 16 mg/L in D. polymorpha. Roundup also caused significant decreases in SYBR-14 staining, significant increases in propidium iodide staining, and significant increases in FITC-PNA staining in D. polymorpha sperm. By not having to rely on egg availability and optimal sperm density, sperm toxicity can be more accurately assessed with flow cytometry as being directly correlated to sperm viability rather than the possibility of altered toxicity results due to sperm-to-egg compatibility.

Feng SM, Zhan WB, Sheng XZ, Yang K, Han JG, Wei JL, Li J, Qiao XT (2009) Response of mucosal and systemic sIgM-positive cells in turbot (Scophthalmus maximus L.) immunization with Edwardsiella tarda. Vet Immunol Immunopathol 129 :108-114


Indirect fluorescence analyze technology (IFAT) and fluorescence-activated cell sorter (FACS) analysis were used to detect surface IgM positive cells (sIgM+ cells) in lymphoid tissues of unvaccinated turbots and vaccinated ones with inactivated Edwardsiella tarda. The results were as follows : (1) The percentage of sIgM+ cells respectively was 0+/-0.00% of skin, 1.58+/-0.40% of gills, 17.05+/-0.39% of peripheral blood (PBL), 21.06+/-1.79% of kidney in unvaccinated healthy turbots (average length : 7.12+/-0.5 cm ; average height : 3.8+/-0.2 cm). (2) After direct immersion (d.i.), the response of sIgM+ cells was produced at first in the gills and thereafter in the kidney and PBL, and a significant increase of the number of sIgM+ cells was found in gills. (3) After immunization via intraperitoneal injection (i.p.), the response of sIgM+ cells was produced simultaneously in the gills, kidney and PBL after i.p., and a significant increase of the number of sIgM+ cells was found in PBL and kidney instead of in gills. These results strongly supported the different immunological role of gills, PBL and kidney upon the different administration routes, and the presence of two compartmental models for immune response in turbot.

Fujii Y, Hiraishi A (2009) Combined Use of Cyanoditolyl Tetrazolium Staining and Flow Cytometry for Detection of Metabolically Active Bacteria in a Fed-batch Composting Process. Microbes and Environments 24 :57-63

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Microbial community dynamics with metabolically active bacteria during the start-up operation of a personal fed-batch composting (FBC) reactor were studied. The FBC reactor was loaded daily with household garbage for 2 months. Metabolically active bacteria were monitored by the redox-dye-staining method using 5-cyano-2,3-ditoryl tetrazolium chloride (CTC), and the fluorescent formazans thus produced were detected by epifluorescence microscopy and flow cytometry (FCM). Microscopic CTC-positive (CTC+) counts accounted for 75-84% of the direct total count during the first week of operation and 19-35% thereafter. Slightly higher CTC+ counts were obtained by FCM. Culture-independent approaches by quinone profiling and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes showed that a drastic population change from ubiquinone-containing members of the Proteobacteria to the Actinobacteria took place during the overall period of operation. The PCR-DGGE analysis of FCM-sorted CTC+ cells supported this observation but gave different major clones from those detected in the total community in some cases. These results suggest that metabolically active bacteria as measured by CTC staining are not always predominant in the FBC process.

Garcia-Garcia E, Garcia-Garcia PL, Rosales C (2009) An fMLP receptor is involved in activation of phagocytosis by hemocytes from specific insect species. Dev Comp Immunol 33 :728-739


In mammalian phagocytes, the bacterial formylated peptide fMLP functions both as a potent enhancer of phagocytosis and chemoattractant. fMLP has been reported to be chemotactic for hemocytes of two marine invertebrates, and of the insect Manduca sexta (Lepidoptera). Whether fMLP is also able to activate phagocytosis has not been explored in hemocytes of any invertebrate. To determine the effect of fMLP on insect hemocyte phagocytosis, in vitro phagocytosis assays were performed with hemocytes from the insects : Gromphadorhina portentosa (Blattodea), Acheta domesticus (Orthoptera), Zophobas morio (Coleoptera), and Galleria mellonella (Lepidoptera). Phagocytosis of latex, zymosan (yeast), Gram-positive and Gram-negative bacteria was measured by flow cytometry, in the presence of increasing fMLP concentrations. G. portentosa hemocytes showed no enhancement of phagocytosis by fMLP. A. domesticus hemocytes had increased phagocytosis of latex and Gram-negative bacteria in the presence of fMLP. Z. morio hemocytes increased phagocytosis of latex, yeast, and Gram-negative bacteria after fMLP stimulation. Galleria mellonella hemocytes increased phagocytosis of latex after fMLP stimulation. Treating hemocytes with Pertussis toxin, a known inhibitor of the signaling pathway initiated by the mammalian fMLP receptor, returned phagocytosis to basal levels. Also, hemocytes from all insect species tested presented a similar chemotactic response to fMLP. These data suggest that, whereas the ability of hemocytes to chemotactically-respond to fMLP is conserved in insects ranging from Blattodea to Lepidoptera, the ability to respond to fMLP by activating phagocytosis is restricted to specific insect species.

Hagedorn M, Ricker J, McCarthy M, Meyers SA, Tiersch TR, Varga ZM, Kleinhans FW (2009) Biophysics of zebrafish (Danio rerio) sperm. Cryobiology 58 :12-19

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In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 +/- 0.02 (SEM), an isosmotic cell volume (V-o) of 12.1 +/- 0.2 mu m(3) (SEM), a water permeability (L-p) in 10% dimethyl sulfoxide of 0.021 +/- 0.001 (SEM) mu m/min/atm, and a cryoprotectant permeability (P-s) of 0.10 +/- 0.01 (SEM) X 10(-3) cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 degrees C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques. Published by Elsevier Inc.

Halloran PR, Rust N, Rickaby REM (2009) Isolating coccoliths from sediment for geochemical analysis. Geochemistry Geophysics Geosystems 10 :-

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Trace element analysis of open-marine sedimentary carbonates provides a wealth of paleoclimate data. At present, the majority of this data is obtained from foraminifera tests. Complications regarding the variability of conditions experienced by foraminifera throughout test formation and the influence of diagenetic processes on sample chemistry limit the value of foraminifera samples in certain situations. Coccoliths, the calcium carbonate plates produced by coccolithophores, represent a second major pelagic open-marine carbonate source with the potential to provide a wide range of valuable trace element proxy data but which have, until now, been unavailable for analysis of many trace elements because of clay contamination. Here we describe a novel technique, which utilizes fast sorting flow cytometry, to enable the production of clay-free sedimentary coccolith samples.

Johnson DR, Czechowska K, Chevre N, van der Meer JR (2009) Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry. Environ Microbiol

Biocides are widely used for domestic hygiene, agricultural and industrial applications. Their widespread use has resulted in their introduction into the environment and raised concerns about potential deleterious effects on aquatic ecosystems. In this study, the toxicity of the biocides triclosan, penconazole and metalaxyl were evaluated with the freshwater bacterium Caulobacter crescentus and with a freshwater microbial community using a combination of single- and double-stain flow cytometric assays. Growth of C. crescentus and the freshwater community were repressed by triclosan but not by penconazole or metalaxyl at concentrations up to 250 muM. The repressive effect of triclosan was dependent on culture conditions. Caulobacter crescentus was more sensitive to triclosan when grown with high glucose at high cell density than when grown directly in sterilized lake water at low cell density. This suggests that the use of conventional growth conditions may overestimate biocide toxicity. Additional experiments showed that the freshwater community was more sensitive to triclosan than C. crescentus, with 10 nM of triclosan being sufficient to repress growth and change the phylogenetic composition of the community. These results demonstrate that isolate-based assays may underestimate biocide toxicity and highlight the importance of assessing toxicity directly on natural microbial communities. Because 10 nM of triclosan is within the range of concentrations observed in freshwater systems, these results also raise concerns about the risk of introducing triclosan into the environment.

Juarez-Jimenez B, Manzanera M, Rodelas B, Martinez-Toledo MV, Gonzalez-Lopez J, Crognale S, Pesciaroli C, Fenice M (2009) Metabolic characterization of a strain (BM90) of Delftia tsuruhatensis showing highly diversified capacity to degrade low molecular weight phenols. Biodegradation

A novel bacterium, strain BM90, previously isolated from Tyrrhenian Sea, was metabolically characterized testing its ability to use 95 different carbon sources by the Biolog system. The bacterium showed a broad capacity to use fatty-, organic- and amino-acids ; on the contrary, its ability to use carbohydrates was extremely scarce. Strain BM90 was identified and affiliated to Delftia tsuruhatensis by molecular techniques based on 16S rRNA gene sequencing. D. tsuruhatensis BM90, cultivated in shaken cultures, was able to grow on various phenolic compounds and to remove them from its cultural broth. The phenols used, chosen for their presence in industrial or agro-industrial effluents, were grouped on the base of their chemical characteristics. These included benzoic acid derivatives, cinnamic acid derivatives, phenolic aldehyde derivatives, acetic acid derivatives and other phenolic compounds such as catechol and p-hydroxyphenylpropionic acid. When all the compounds (24) were gathered in the same medium (total concentration : 500 mg/l), BM90 caused the complete depletion of 18 phenols and the partial removal of two others. Only four phenolic compounds were not removed. Flow cytometry studies were carried out to understand the physiological state of BM90 cells in presence of the above phenols in various conditions. At the concentrations tested, a certain toxic effect was exerted only by the four compounds that were not metabolized by the bacterium.

Kang H, Sanchez Alvarado A (2009) Flow cytometry methods for the study of cell-cycle parameters of planarian stem cells. Dev Dyn 238 :1111-1117


Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo.

Levy JL, Stauber JL, Wakelin SA, Jolley DF (2009) The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays. Chemosphere 74 :1266-1274

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Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each ; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchnerielia subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, A subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46 mu g Cu L-1, while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208 mu g Cu L-1. However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The "axenic" cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment. 0 2008 Elsevier Ltd. All rights reserved.

Lopez-Flores R, Boix D, Badosa A, Brucet S, Quintana XD (2009) Environmental factors affecting bacterioplankton and phytoplankton dynamics in confined Mediterranean salt marshes (NE Spain). Journal of Experimental Marine Biology and Ecology 369 :118-126

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Environmental factors accountable for bacterioplankton or phytoplankton biomass dominance were analysed in a confined Mediterranean salt marsh (Emporda Wetlands, NE Spain). Two basins located in the same salt marsh, and with differences in size and catchment’s area were compared, during four characteristic situations of the hydroperiod. Since bacterio- or phytoplankton relationships may be affected by other factors such as diel variations or vertical differences in nutrient composition and distribution, high frequency fluctuations due to these factors were also taken into account. Differences in catchment area appeared to be the more plausible explanation of differences in nutrient and organic carbon accumulation among basins, since during confinement basins essentially accumulate the allochthonous nutrient and organic matter supplies that previously entered by runoff. DOC (Dissolved Organic Carbon) favoured the bacterioplankton biomass increase, but also was the main variable significantly affecting phytoplankton biomass. Basins showed marked differences in bacterio- and phytoplankton dominances. Relationships between phytoplankton and bacterioplankton were positive, negative or not significant, depending on the basin and on the period of the year. The phytoplankton mixotrophic capabilities, both phagotrophy and osmotrophy, and their production of UV-screening compounds, as sunscreen, may explain the significant correlation between DOC and phytoplankton biomass, and the significant effect of phytoplankton on bacterioplankton found in these ecosystems. (C) 2008 Elsevier B.V. All rights reserved.

Loureiro S, Jauzein C, Garces E, Collos Y, Camp J, Vaque D (2009) The significance of organic nutrients in the nutrition of Pseudo-nitzschia delicatissima (Bacillariophyceae). Journal of Plankton Research 31 :399-410

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The influence of organic nutrients on the evolution of Pseudo-nitzschia delicatissima cultures was investigated in an enrichment experiment with high-molecular-weight dissolved organic matter (HMWDOM) and in an uptake assay with N-15-ammonium and N-15-urea. HMWDOM was extracted from seawater collected at a nearby shore station during the decline of a diatom bloom. Four incubations were prepared : L1/5+DOM (P. delicatissima grown in L1 growth medium with 1/5 of the nitrate concentration of standard L1), (L1-N)+DOM (L1 without nitrate, i.e. nitrogen-deficient treatment), L1-DOM (control culture without added DOM) and BV+DOM (bacterial and viral control, free of microalgae). Incubations were carried out for 10 days. Chlorophyll a concentrations differed after day 4 and reached higher levels in the L1-DOM incubation by the end of the experiment ; however, similar growth rates were observed in all incubations (1.64 +/- 0.05 divisions day(-1)). The persistently lower cellular chlorophyll content in (L1-N)+DOM during the experiment was consistent with N limitation conditions. The data suggested that the nitrogen needed for the growth of (L1-N)+DOM cells might have originated from the DOM. Based on the results of N-15 uptake assays, it was concluded that P. delicatissima more readily acquires ammonium than urea. Nevertheless, under low N conditions, P. delicatissima may use urea as an alternative N source, and comparable photosynthetic rates are attained on either substrate. Taken together, our results suggest a positive effect of organic nutrients on the growth of P. delicatissima.

Ma Y, Zeng Y, Jiao N, Shi Y, Hong N (2009) Vertical distribution and phylogenetic composition of bacteria in the Eastern Tropical North Pacific Ocean. Microbiol Res 164 :624-633


The vertical community structure of bacteria along a depth profile in the Eastern Tropical North Pacific Ocean (13 degrees N, 104 degrees W) was studied by flow cytometry measurement and 16S rRNA gene clone libraries analysis. Picoeukaryotes and Synechococcus peaked at 30 m and decreased sharply below 50 m, while Prochlorococcus peaked at both 30 and 100 m layers and disappeared below 200 m. Heterotrophic bacteria peaked above shallow thermocline and decreased along the depth profile. Sequences of total 322 clones from four clone libraries (10, 100, 1000, and 3000 m) clustered into nine major lineages. gamma-Proteobacteria dominated all the depths and occupied almost the whole bacterial community at the 3000 m. alpha-Proteobacteria was abundant throughout the water column except near the sea bottom, and delta-Proteobacteria peaked at the 1000 m depth. Cyanobacteria were primarily limited to the photic zone, and the genetic diversity of Prochlorococcus showed a good correlation with niche adaptation. The appearance of the Cytophaga-Flexibacter-Bacteroides (CFB) group did not show a clear relationship with depth. Actinobacteria were found both in the photic zone and in deep water. Planctomyetes, Acidobacteria, and Verrucomicrobia were present as minor groups and more dominant in the deeper layers of water.

Malits A, Weinbauer MG (2009) Effect of turbulence and viruses on prokaryotic cell size, production and diversity. Aquatic Microbial Ecology 54 :243-254

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A factorial design was used to assess the roles of turbulence and viral infection in prokaryotic production and diversity in a spring phytoplankton bloom experiment in the Bay of Ville-franche, France, Several consistent trends were observed in 2 experiments : (1) turbulence stimulated prokaryotic production, (2) prokaryotic cell length increased in experimental turbulence and virus treatments, and (3) organic micro-aggregates with attached prokaryotes formed only in the turbulence treatments and seemed to be reduced in the presence of viruses. We conclude that turbulence likely influenced prokaryotes indirectly by affecting micro-aggregate formation and nutrient availability, Turbulence and viruses had only small influences on the number of bacterial and archaeal bands detected by 16S rRNA gene denaturing gradient gel electrophoresis. However, taking into account presence versus absence of specific bands and their intensities, we detected strong effects in the experiments. We not only detected a negative effect of viruses, but also found that some bands increased in intensity in the presence of active viruses, e.g. one of 3 phylotypes affiliated with the Rhodobacteriaceae. In both experiments, several consistent patterns were found : (1) a phylotype affiliated with Roseobacter was negatively affected (in terms of band intensity) by viruses and turbulence, (2) the relative band intensity of a Rhodobacter increased in the turbulence treatments, and (3) a phylotype related to Oceanospirillum was detected only in the turbulence treatment. We suggest that turbulence and viruses play a significant and previously neglected role in shaping prokaryotic diversity, aggregation and production.

Matson CW, Gillespie AM, McCarthy C, McDonald TJ, Bickham JW, Sullivan R, Donnelly KC (2009) Wildlife toxicology : biomarkers of genotoxic exposures at a hazardous waste site. Ecotoxicology 18 :886-898


A large number of hazardous waste sites in the United States have undergone the initial stages of remediation or containment. At many of the remaining sites, the potential for exposure to ecological receptors is a primary concern. This manuscript reports on studies to investigate the impact on ecological receptors exposed to complex mixtures at a former creosote facility. Currently there are isolated areas on-site that were not addressed in the initial removal action that appear to be releasing polycyclic aromatic hydrocarbons (PAHs) to the surrounding environment. The U.S. EPA collected environmental samples and performed ex situ sediment bioassays to measure chronic toxicity ; whereas, this study describes an in situ study to measure biomarkers of effect in two ecological receptors. Mosquitofish (Gambusia affinis) and cricket frogs (Acris crepitans) were collected from a small intermittent creek adjacent to the site, and reference stations. A weight-of-evidence ecological risk assessment was completed for the amphibian and fish communities. The ecological risk assessment was developed using analysis of media chemistry, body burden of specific PAHs, bioassay results, community surveys, and cellular genome size variation as a biomarker of genotoxicity. Flow cytometric estimates of chromosomal damage were significantly elevated for both mosquitofish and cricket frogs inhabiting the contaminated site, relative to at least one reference site. Surface water screening values for fish and amphibians exceeded screening values for PAHs by more than one order of magnitude in the on-site creek, and sediment PAH concentrations were extremely high (up to 1,549 microg/dry g). Tissue concentrations of PAHs were below screening values. Media chemistry, bioassay and genotoxicity data all support the same conclusion that on-site PAHs continue to impact aquatic receptors. The genotoxicity findings are consistent with and contribute to results of the weight-of-evidence ecological risk assessment. The results support continuing efforts to incorporate biomarkers as valuable lines of evidence within ecological risk assessment.

Mitbavkar S, Saino T, Horimoto N, Kanda J, Ishimaru T (2009) Role of environment and hydrography in determining the picoplankton community structure of Sagami Bay, Japan. Journal of Oceanography 65 :195-208

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Seasonal variations in the picoplankton community were investigated from June 2002 to March 2004 within the photic zone of Sagami Bay, Japan. The study area was mostly dominated by coastal waters during the warm period (mixed layer water temperature a parts per thousand yen 18A degrees C). During the cold period (mixed layer water temperature a parts per thousand currency sign 18A degrees C), the water mass was characterized by low temperature and high saline waters indicative of the North Pacific Subtropical Mode Water (NPSTMW). Occasionally, a third type of water mass characterized by high temperature and low saline properties was observed, which could be evidence of the intrusion of warm Kuroshio waters. Synechococcus was the dominant picophytoplankton (5-28 x 10(11) cells m(-2)) followed by Prochlorococcus (1-5 x 10(11) cells m(-2)) and picoeukaryotes during the warm period. Heterotrophic bacteria dominated the picoplankton community throughout the year, especially in the warm period. During the Kuroshio Current advection, cyanobacterial abundance was high whereas that of picoeukaryotes and heterotrophic bacteria was low. During the cold period, homogeneously distributed, lower picophytoplankton cell densities were observed. The dominance of Synechococcus in the warm period reflects the importance of high temperature, low salinity and high Photosynthetically Active Radiation (PAR) on its distribution. Cyanobacterial and heterotrophic bacterial abundance showed a positive correlation with temperature. Prochlorococcus and picoeukaryotes showed a positive correlation with nutrients. Picoeukaryotes were the major contributors to the picophytoplankton carbon biomass. The annual picophytoplankton contribution to the photosynthetic biomass was 32 +/- 4%. These observations suggest that the environmental conditions, combined with the seasonal variability in the source of the water mass, determines the community structure of picoplankton, which contributes substantially to the phytoplankton biomass and can play a very important role in the food web dynamics of Sagami Bay.

Moran XAG, Calvo-Diaz A (2009) Single-cell vs. bulk activity properties of coastal bacterioplankton over an annual cycle in a temperate ecosystem. Fems Microbiology Ecology 67 :43-56

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The connections between single-cell activity properties of heterotrophic planktonic bacteria and whole community metabolism are still poorly understood. Here, we show flow cytometry single-cell analysis of membrane-intact (live), high nucleic acid (HNA) content and actively respiring (CTC+) bacteria with samples collected monthly during 2006 in northern Spain coastal waters. Bulk activity was assessed by measuring H-3-Leucine incorporation and specific growth rates. Consistently, different single-cell relative abundances were found, with 60-100% for live, 30-84% for HNA and 0.2-12% for CTC+ cells. Leucine incorporation rates (2-153 pmol L-1 h(-1)), specific growth rates (0.01-0.29 day(-1)) and the total and relative abundances of the three single-cell groups showed marked seasonal patterns. Distinct depth distributions during summer stratification and different relations with temperature, chlorophyll and bacterial biovolume suggest the existence of different controlling factors on each single-cell property. Pooled leucine incorporation rates were similarly correlated with the abundance of all physiological groups, while specific growth rates were only substantially explained by the percentage of CTC+ cells. However, the ability to reduce CTC proved notably better than the other two single-cell properties at predicting bacterial bulk rates within seasons, suggesting a tight linkage between bacterial individual respiration and biomass production at the community level.

Morgan DA, Class R, Violetta G, Soslau G (2009) Cytokine mediated proliferation of cultured sea turtle blood cells : morphologic and functional comparison to human blood cells. Tissue Cell 41 :299-309


Blood cells from three different sea turtle species were cultured for approximately 3 weeks in nutrient medium supplemented with recombinant human cytokines known to induce terminal maturation of human hematological stem cells. Cultured turtle erythrocytes were translucent, approximately 10x larger than human erythrocytes, contained a single fluorescent inclusion body, contained nuclear epsilon (embryonic) globin proteins, and, absent of organelles while fresh cells contained few, but well defined mitochondria. Cells with basophilic cytoplasm and in all stages of proliferation were observed in cytokine-supplemented cultures and appeared to possess active protein synthesis. Cultured thrombocytes aggregated in response to agonists for at least 8 days, post-collection, contained P-selectin in the nucleus of 6 day cultured cells which appeared to be released after activation with collagen, and after 6 days had no organelles or open canalicular-like system (OCS) while freshly isolated cells demonstrated few, if any organelles but had a well developed OCS. The response of turtle cells to apparently homologous but unnatural human cytokines and the sustained biological properties of thrombocytes identify this suspension culture system as a powerful tool to explore the evolution of cell types and molecular components of hematopoiesis and hemostasis.

O’Brien JK, Steinman KJ, Robeck TR (2009) Application of sperm sorting and associated reproductive technology for wildlife management and conservation. Theriogenology 71 :98-107


Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.

Palenik B, Ren Q, Tai V, Paulsen IT (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Environ Microbiol 11 :349-359


The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.

Posmyk MM, Balabusta M, Wieczorek M, Sliwinska E, Janas KM (2009) Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. Journal of Pineal Research 46 :214-223

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The relationship between germination and melatonin applied during osmo- and hydropriming was studied in cucumber seeds. The proportion of nuclei with different DNA contents, the mean ploidy and the (2C + 4C = 8C)/2C ratio in unprimed and primed, dry and imbibed at 10 degrees C seeds were established by flow cytometry. Thiobarbituric acid reactive substances and protein oxidation were also estimated. Melatonin and indole-3-acetic acid (IAA) concentrations in the seeds were determined using high-performance liquid chromatography with electrochemical detection. Being sensitive to chilling stress, seeds that germinated well (99%) at 25 degrees C showed only 30% germination at 15 degrees C, and almost no germination (4%) at 10 degrees C. Hydropriming in water improved seed germination to 50-60% at 15 degrees C and the addition of melatonin (25-100 m) also increased the rate of germination. Osmopriming in polyethylene glycol increased germination at 15 degrees C to 78%, and 98% when combined with 50 m melatonin. Osmoprimed seeds germinated even at 10 degrees C and reached 43%, and 83% when 50 m melatonin was applied. None of the treatments induced DNA synthesis, although during the first 24 hr of imbibition at 10 degrees C the mean ploidy and the (2C + 4C = 8C)/2C ratio increased, which is indicative of the advanced Phase II of germination. Hydro- and osmopriming slightly decreased IAA content in the seeds in most of the cases ; only hydropriming with 100 and 500 m melatonin increased it. Melatonin protected membrane structure against peroxidation during chilling, but excessive melatonin levels in cucumber seeds (similar to 4 mu g/g fresh weight) provoked oxidative changes in proteins. There is still lack of information explained clearly the role of melatonin in plant physiology. This molecule acts multidirectionally and usually is alliged to other compounds.

Prado R, Garcia R, Rioboo C, Herrero C, Abalde J, Cid A (2009) Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat. Environ Int 35 :240-247


The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Toxic effects of these pollutants on microalgae are generally evaluated using phytotoxicity tests based on growth inhibition, a population-based parameter. However, physiological cellular endpoints could allow early detection of cell stress and elucidate underlying toxicity mechanisms. Effects of the herbicide paraquat on the freshwater microalga Chlamydomonas moewusii were studied to evaluate growth rate and cellular parameters such as cellular viability and metabolic activity assayed by flow cytometry and DNA damage assayed by the comet assay. Sensitivity of growth and parameters assayed by flow cytometry were similar, showing a significant effect in cultures exposed to a paraquat concentration of 0.1 microM or higher, although in cultures exposed during 48 h to 0.05 microM, a significant stimulation of cellular fluorescein fluorescence was observed, related to cellular metabolic activity. After only 24 h of herbicide exposure significant DNA damage was observed in microalgal cells exposed to all paraquat concentrations assayed, with a 23.67% of comets in cultures exposed to 0.05 microM, revealing the genotoxicity of this herbicide. Taking into account the results obtained, comet assay provides a sensitive and rapid system for measuring primary DNA damage in Chlamydomonas moewusii, which could be an important aspect of environmental genotoxicity monitoring in surface waters.

Rioboo C, O’Connor JE, Prado R, Herrero C, Cid A (2009) Cell proliferation alterations in Chlorella cells under stress conditions. Aquat Toxicol 94 :229-237


Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used : (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and reproduction at microalgal cell level.

Rivers AR, Jakuba RW, Webb EA (2009) Iron stress genes in marine Synechococcus and the development of a flow cytometric iron stress assay. Environ Microbiol 11 :382-396


Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe(3+) transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5-54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.

Rodriguez-Blanco A, Ghiglione JF, Catala P, Casamayor EO, Lebaron P (2009) Spatial comparison of total vs. active bacterial populations by coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea. FEMS Microbiol Ecol 67 :30-42


Spatial distributions of both total (i.e. 16S rDNA-based fingerprints) and active (i.e. 16S rRNA-based fingerprints) bacterial populations, together with total bacterial activity measured by 3H-leucine incorporation, were studied along a 98 km transect in the NW Mediterranean Sea. Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting was coupled to a clone library, allowing CE-SSCP peaks identification and the monitoring of the spatial variation of bacterial phylotypes. Up to 80% of the community peaks matched those obtained from clone library sequences, accounting for 86.7% of the total fingerprinting area. A good agreement was found between the relative abundance of Prochlorococcus in the CE-SSCP fingerprints and flow cytometry counts (r2=0.66, P<0.05). The largest differences between total and active bacterial populations distribution were found at depths with higher bacterial activity (i.e. surface and deep chlorophyll maximum, DCM). SAR11 at the surface and Gammaproteobacteria at the DCM were the most abundant groups on the 16S rDNA-based fingerprints. However, their ratio of relative importance between rRNA : rDNA was <1> 1 both at the surface and at the DCM. These results emphasize the need for combining rDNA- and rRNA-based analyses to better understand the functional role of individual bacterial populations in situ.

Rohacova J, Marin ML, Martinez-Romero A, Diaz L, O’Connor JE, Gomez-Lechon MJ, Donato MT, Castell JV, Miranda MA (2009) Fluorescent benzofurazan-cholic acid conjugates for in vitro assessment of bile acid uptake and its modulation by drugs. ChemMedChem 4 :466-472


One of the most common mechanisms of hepatotoxicity is drug-induced cholestasis. Hence, new approaches for screening the cholestatic potential of drug candidates are desirable. In this context, we describe herein the use of synthetic 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent conjugates of cholic acid (ChA) at positions 3alpha, 3beta, 7alpha, and 7beta for in vitro assessment of bile acid uptake. All the conjugates show a strong absorption band between 400 and 550 nm and have a fluorescence quantum yield of approximately 0.45, with an emission maximum centered at approximately 530 nm. After their photophysical characterization, 3alpha-, 3beta-, 7alpha-, and 7beta-NBD-ChA were used to monitor uptake in freshly isolated rat hepatocytes by means of a previously optimized flow cytometry technique. Transport of the cholic acid derivatives inside the cell was detected and quantified by measuring the increase of NBD green fluorescence within cells over time. The effect of troglitazone, a well-known inhibitor of bile acid uptake by the sodium taurocholate co-transporting polypeptide, supports the specificity of fluorescent NBD-ChA transport. According to the final intracellular fluorescence level attained and the uptake rate, 3alpha-NBD-ChA was found to be the most efficient derivative. Furthermore, sodium valproate, cyclosporin A, and chlorpromazine decreased the uptake of 3alpha-NBD-ChA, in agreement with their relative in vivo potency as cholestatic compounds ; in contrast, sodium citrate (the negative control) had no effect. These results support the suitability of the in vitro flow cytometric assay with NBD-ChA to detect compounds that affect bile acid uptake.

Rohacova J, Marin ML, Martinez-Romero A, O’Connor JE, Gomez-Lechon MJ, Donato MT, Castell JV, Miranda MA (2009) Synthesis of new, UV-photoactive dansyl derivatives for flow cytometric studies on bile acid uptake. Org Biomol Chem 7 :4973-4980


Four new fluorescent derivatives of cholic acid have been synthesized ; they incorporate a dansyl moiety at 3alpha-, 3beta-, 7alpha- or 7beta- positions. These cholic acid analogs are UV photoactive and also exhibit green fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile acid transport by flow cytometry.

Rose JM, Vora NM, Countway PD, Gast RJ, Caron DA (2009) Effects of temperature on growth rate and gross growth efficiency of an Antarctic bacterivorous protist. Isme Journal 3 :252-260

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The effects of temperature on the growth rate and gross growth efficiency (GGE) of the heterotrophic nanoflagellate, Paraphysomonas imperforata, cultured from the Ross Sea, Antarctica were investigated using five experimental temperatures (range 0-20 degrees C). This bacterivorous protist exhibited measurable growth over the temperature range examined, although temperature exerted a significant effect on its growth rate. There was no evidence for an effect of temperature on GGE. The growth rates and GGE of our Antarctic P. imperforata isolate were compared to values reported for other cultures of species from this genus. A wide range of growth efficiencies have been reported for different strains of Paraphysomonas spp., but our estimates were comparable to mean/median values reported in the literature. The growth rates of our Antarctic P. imperforata were similar to rates obtained for an Arctic conspecific at low temperatures (0-5 degrees C), among the highest reported rates for any Paraphysomonas species at intermediate temperatures (10-15 degrees C) and similar to rates reported for temperate congeners and conspecifics at 20 degrees C. Q(10) values of 15, 2.2, 3.6 and 0.93 were calculated for growth rates at 5 degrees C intervals between 0 and 20 degrees C, respectively. Results indicated that our Antarctic P. imperforata grew at rates comparable to other polar isolates at ambient polar temperatures, but these low temperatures may be outside the physiological optimum for the isolate.

Rossi S, Sa-Rocha VM, Kinoshita D, Genoy-Puerto A, Zwarg T, Werneck MR, Sa-Rocha LC, Matushima ER (2009) Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae). Braz J Biol 69 :899-905


Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the Sao Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences. The following stimuli were applied in the assessment of leukocyte function : Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles, Alexa Fluor 594 conjugate for phagocytosis evaluation. Three cell populations were identified : heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Saison C, Perreault F, Daigle JC, Fortin C, Claverie J, Morin M, Popovic R (2009) Effect of core-shell copper oxide nanoparticles on cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, Chlamydomonas reinhardtii. Aquat Toxicol

The effect of core-shell copper oxide nanoparticles with sizes smaller than 100nm on cellular systems is still not well understood. Documenting these effects is pressing since core-shell copper oxide nanoparticles are currently components of pigments used frequently as antifouling paint protecting boats from crustacean, weed and slime fouling. However, the use of such paints may induce strong deteriorative effects on different aquatic trophic levels that are not the intended targets. Here, the toxic effect of core-shell copper oxide nanoparticles on the green alga, Chlamydomonas reinhardtii was investigated with regards to the change of algal cellular population structure, primary photochemistry of photosystem II and reactive oxygen species formation. Algal cultures were exposed to 0.004, 0.01 and 0.02g/l of core-shell copper oxide nanoparticles for 6h and a change in algal population structure was observed, while the formation of reactive oxygen species was determined using the 2’,7’-dichlorodihydrofluorescein diacetate marker measured by flow cytometry. For the study of the photosystem II primary photochemistry we investigated the change in chlorophyll a rapid rise of fluorescence. We found that core-shell copper oxide nanoparticles induced cellular aggregation processes and had a deteriorative effect on chlorophyll by inducing the photoinhibition of photosystem II. The inhibition of photosynthetic electron transport induced a strong energy dissipation process via non-photochemical pathways. The deterioration of photosynthesis was interpreted as being caused by the formation of reactive oxygen species induced by core-shell copper oxide nanoparticles. However, no formation of reactive oxygen species was observed when C. reinhardtii was exposed to the core without the shell or to the shell only.

Shapiro K, Mazet JA, Schriewer A, Wuertz S, Fritz H, Miller WA, Largier J, Conrad PA (2009) Detection of Toxoplasma gondii oocysts and surrogate microspheres in water using ultrafiltration and capsule filtration. Water Res

While reports on waterborne infections with Toxoplasma gondii are emerging worldwide, detection of this zoonotic parasite in water remains challenging. Lack of standardized and quantitative methods for detection of T. gondii oocysts in water also limits research on the transport and fate of this pathogen through aquatic habitats. Here, we compare the ability of hollow-fiber ultrafiltration and capsule filtration to concentrate oocysts in spiked tap water, fresh surface water, and seawater samples. Detection of T. gondii oocysts in concentrated samples was achieved using molecular methods, as well as visually via epifluorescent microscopy. In addition to oocysts, water samples were spiked with T. gondii surrogate microspheres, and detection of microspheres was performed using flow cytometry and epifluorescent microscopy. Results demonstrate that both water concentration methods followed by microscopy allowed for quantitative detection of T. gondii oocysts and surrogate microspheres. For T. gondii oocysts, microscopy was more sensitive than TaqMan and conventional PCR, and allowed for detection of oocysts in all water samples tested. Compared with flow cytometry, microscopy was also a more cost-efficient and precise method for detection of fluorescent surrogate microspheres in tap, fresh and seawater samples. This study describes a novel approach for quantitative detection of T. gondii oocysts in drinking and environmental water samples. The techniques described for concentrating and detecting surrogate microspheres have broad application for evaluating the transport and fate of oocysts, as well as the efficiency of water treatment methods for removal of T. gondii from water supplies.

Shelley LK, Balfry SK, Ross PS, Kennedy CJ (2009) Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 92 :95-103


Many current-use pesticides (CUPs) are found at increasing concentrations in aquatic environments, yet relatively little is known about their effects on the immune system of fish. We examined the in vivo effects of three pesticides (chlorothalonil, cypermethrin and pentachlorophenol) on the immune system of juvenile rainbow trout (Oncorhynchus mykiss) by assessing a suite of innate immune function tests, as well as a host resistance test using Listonella anguillarum. Increased activity of phagocytic leukocytes, as evidenced using flow cytometry, was observed following 28-day exposures to pentachlorophenol (1 microg/L and 2 microg/L), but not for cypermethrin or chlorothalonil, although a trend of increasing activity was noted for chlorothalonil. No recovery was observed during a 14-day post-exposure chlorothalonil experiment, as evidenced by continued elevation of respiratory burst and percent phagocytic cells at the lowest exposure concentrations (100 ng/L and 200 ng/L), suggesting a prolonged CUP-induced impact on the immune system. No effects of any pesticide on body weights, direct lethality, serum lysozyme or relative leukocyte differential were observed, suggesting that modulation of the cellular responses of the innate immune system represents a sensitive sub-lethal endpoint for these three pesticides. However, a lack of detectable effects of these CUPs on host resistance to L. anguillarum in our study may reflect a dose-response range that did not elicit an effect on those immune responses responsible for control and clearance of this particular pathogen. Additional research may provide more insight into the immunomodulatory effects of these and other CUPs, and the implications for host resistance against a variety of bacterial, viral and macroparasitic pathogens.

Silva R, Negri R, Lutz V (2009) Summer succession of ultraphytoplankton at the EPEA coastal station (Northern Argentina). Journal of Plankton Research 31 :447-458

<Go to ISI> ://000263962900008

Ultraphytoplankton (< 5 mu m) are important members of food webs in any pelagic system. Temporal succession in their community structure was studied at a coastal station (38 degrees 28’S-57 degrees 41’W), "Estacion Permanente de Estudios Ambientales (EPEA)", during summer 2001-2002. Prokaryotic and eukaryotic algae of this fraction, and small protozoa were identified, enumerated and optically characterized using a combination of methods : classic and fluorescence microscopy, flow cytometry and absorption spectra. Ultraphytoplankton contributed up to 90% of total chlorophyll a during this summer. Synechococcus dominated the community and their highest biomass was related to high temperature and stability during January. Chlorophyta coccal algae had a maximum biomass in February, when the water column was weakly stratified. Cryptophyceae, Prymnesiophyceae, Prasinophyceae, Bacillariophyceae and Chrysophyceae ultra-algae were present in variable proportions throughout the period. Variations in optical properties were significantly related to the shift from prokaryotic to eukaryotic coccal cells, which would allow the possibility of inferring phytoplankton types bio-optically. The ultraphytoplankton succession was mainly driven by physical forces, especially the degree of stability. Algae with coccal life-form strategies seem to be a major source of biogenic carbon supporting the functioning of this pelagic coastal ecosystem during summer.

Stachowski-Haberkorn S, Quiniou L, Beker B, Haberkorn H, Marie D, de la Broise D (2009) Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities. Ecotoxicology 18 :364-376


The impacts of the fungicide Opus (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg l(-1), TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 microg l(-1) of epoxiconazole. When exposed to 100 microg l(-1), all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities.

Tadonleke RD, Leberre B, Perreau F, Humbert JF (2009) Responses of lake bacterioplankton activities and composition to the herbicide diuron. Aquat Toxicol 94 :103-113


The direct effects of pesticides on aquatic bacteria are poorly known. We experimentally investigated the direct effects of diuron (herbicide) on the composition and activities of lake bacterioplankton, using Denaturing Gradient Gel Electrophoresis (DGGE), cloning/sequencing, and flow cytometry with dyes that allow detection of dead cells, cells with depolarized membranes and cells with esterase activity (for physiological state). Generally, diuron had negative impacts on bacterial viability and abundance. Bacterial production strongly correlated with ammonium in controls, but not in diuron-treated samples. Moreover the increase in nitrate concentration with the proportion of dead bacteria was significantly higher in controls, providing evidence not previously shown for natural communities, that diuron may alter the mineralization of organic matter and nitrification. A picocyanobacteria and members of the family Flavobacteriaceae, known to degrade complex polymeric organic matter in aquatic systems were negatively affected by diuron. Except that, the DGGE banding patterns in controls and in polluted samples were generally similar, suggesting no perceptible susceptibility of major bacterial groups, and contrasting with previous reports that diuron has a strong impact on bacterial community composition. Our data suggest that diuron may affect functioning of aquatic systems through negative impacts on some bacterial phylotypes and bacterial cycling of nitrogen.

Tang X, Zhan W, Sheng X, Chi H (2009) Immune response of Japanese flounder Paralichthys olivaceus to outer membrane protein of Edwardsiella tarda. Fish Shellfish Immunol

The humoral and cellular immune responses of Japanese flounder, Paralichthys olivaceus, were investigated following intraperitioneal injection with outer membrane protein (OMP) of Edwardsiella tarda in Freund’s incomplete adjuvant (FIA). The specific serum antibody titer against OMP of E. tarda were measured using ELISA for 14 weeks, and the total serum antibody concentrations were also determined according to the sandwich ELISA standard model constructed using purified IgM. Both of the specific and total antibodies had an increase and reached their peaks 4 weeks after immunization. Simultaneously, the percentages of sIg + lymphocytes in blood, spleen, pronephros and mesonephros were detected by flow cytometry. It was shown that the percentages of sIg + lymphocytes in all lymphoid organs reached their peak levels 4 weeks after immunization, and then decreased gradually. To investigate the protection against infection, three challenges were performed in the same way at day 14, 30 and 100 after immunization, fish challenged at day 30 showed a higher relative percentage survival (RPS) of 71 compared to the 14-day group (30) and 100-day group (53), which indicated a positive correlation between the survival and the levels of the antibody.

Wang Y, Hammes F, Boon N, Chami M, Egli T (2009) Isolation and characterization of low nucleic acid (LNA)-content bacteria. Isme J 3 :889-902


Most planktonic bacteria are ’uncultivable’ with conventional methods. Flow cytometry (FCM) is one approach that has been taken to study these bacteria. In natural aquatic environments, bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed with FCM after staining with fluorescent dyes. Although several studies have focused on the relative abundance and in situ activities of these two groups, knowledge on the growth of particularly LNA bacteria is largely limited. In this study, typical LNA bacteria were enriched from three different freshwater sources using extinction dilution (ED) and fluorescence-activated cell sorting (FACS). We have shown for the first time that LNA bacteria can be isolated and cultivated by using sterile freshwater as a growth medium. During growth, the typical LNA characteristics (that is, low-fluorescence intensity and sideward scatter (SSC)) remained distinct from those of typical HNA bacteria. Three LNA pure cultures that are closely affiliated to the Polynucleobacter cluster according to 16S rRNA sequencing results were isolated. Owing to their small size, cells of the isolates remained intact during cryo-transmission electronic microscopy examination and showed a Gram-negative cell-wall structure. The extremely small cell volume (0.05 microm3) observed for all three isolates indicates that they are among the smallest free-living heterotrophic organisms known in culture. Their isolation and cultivation allow further detailed investigation of this group of organisms under defined laboratory conditions.

Yano A, von Schalburg K, Cooper G, Koop BF, Yoshizaki G (2009) Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss). Mol Reprod Dev 76 :246-254


The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.

Zhang X, Hu HY, Men YJ, Yang J, Christoffersen K (2009) Feeding characteristics of a golden alga (Poterioochromonas sp.) grazing on toxic cyanobacterium Microcystis aeruginosa. Water Res 43 :2953-2960


Microcystis aeruginosa has quickly risen in infamy as one of the most universal and toxic bloom-forming cyanobacteria. Here we presented a species of golden alga (Poterioochromonas sp. strain ZX1), which can feed on toxic M. aeruginosa without any adverse effects from the cyanotoxins. Using flow cytometry, the ingestion and maximal digestion rates were estimated to be 0.2 approximately 1.2 and 0.2 M. aeruginosa cells (ZX1 cell)(-1)h(-1), respectively. M. aeruginosa in densities below 10(7)cells mL(-1) could be grazed down by ZX1, but no significant decrease was observed when the initial density was 3.2 x 10(7)cells mL(-1). ZX1 grazing was a little influenced by the light intensity (0.5 approximately 2500l x) and initial pH of the medium (pH=5.0 approximately 9.5). ZX1 could not survive in continuous darkness for longer than 10 days. The pH value was adjusted to 8 by ZX1 while to 10 by M. aeruginosa. This study may shed light on understanding the ecological interactions between M. aeruginosa and mixotrophic Poterioochromonas sp. in aquatic ecosystems.