Flow cytometry and methods to count aquatic viruses and assess viral-induced induced mortality of bacteria

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Domaizon I 1 & Jacquet S 1 (1) UMR CARRTEL, Equipe de Microbiologie Aquatique, Thonon &

1 Viruses Bacteria Flow cytometry and methods to count aquatic viruses and assess viral-induced induced mortality of bacteria Personnic S 1, Duhamel S 1, Sime-Ngando T 2, Domaizon I 1 & Jacquet S 1 (1) UMR CARRTEL, Equipe de Microbiologie Aquatique, Thonon & Bourget-du-lac, France (2) Laboratoire de Biologie des Protistes, Campus des Cézeaux,Aubière, France

2 Content Viruses: who are you? The aquatic microbial network Viral roles Methods to count aquatic viruses EFM TEM FCM Comparison EFM / TEM / FCM Case studies dealing with the viral community dynamics Cases studies dealing with the viral role as mortality agents Conclusion

3 The aquatic viruses Are you numerous? -10 to 100 more abundant than heterotrophic bacteria - About 10 5 to 10 8 particles per ml of water, the most abundant aquatic biological entities - Abundances: Viruses > Heter. Bacteria > Cyanobacteria > Pico-Nano eukaryotes Who are you? -Size: 20 to 200 nm (most < 60 nm) - Diversity? Poorly known - Mortality agents, obligatory parasites - Ds DNA containing particles Fig: Schema of a virus T4 Are you fundamental to understand microbial ecology?

4 The aquatic food web DOM allochtonous Microbial loop Phytoplankton DOM Heterotrophic Bacteria Zooplankton Protistes flagellates Protistes ciliates Higher Predators

5 Viruses DOM allochtonous DOM allochtone Microbial loop Phytoplankton DOM DOM Heterotrophic Bacteria Heterotrophic Bacteria Zooplankton Viruses Viruses Protistes flagellés Protistes Protistes ciliés flagellates Protistes ciliates Higher Predators Prédators

6 The role of viruses! Regulating factor of microbial communities abundance ;! Regulating factor of bacterial diversity ;! Responsible for bacterial gene transfer (transduction).. Understanding relations between microbial communities without viruses Not possible. Analysing and explaining bacterial diversity without viruses? Not possible Need to analyze viral communities

7 Methods to count aquatic viruses There are 3 principal techniques to count viruses in the field of aquatic sciences. EFM EpiFluorescence Microscopy. TEM Transmission Electron Microscopy. FCM Flow Cytometry Note that other techniques exist to enumerate viruses

8 EFM: Epifluorescence microscopy Viruses Bacteria Type: Microscopy Principle: Target bacterial and viral DNA fluorescence after light excitation. DNA fluorescence is obtained using a highly fluorescent acid-nucleic dye (SYBR Gold for example). Counts: human-eye

9 Advantages See (pretend to see) the organisms Relatively quick method Disadvantages Human counts (reproducibility?) Particle Fluorescence (fading) Conversion of the viruses really counted to viruses per ml

$Diffraction of an electron$ flow by colored bacterial and

10 TEM: Transmission electron microscopy Viruses Bacteria Type: Microscopy Principle: Diffraction of an electron flow by colored bacterial and viral cell compounds Counts: human-eye

11 Advantages See the organisms Access of different parameters: counts but also BS, FVIC (+FIC, VIBM) Disadvantages Human counts (reproducibility?) Impossible to use it for routine quantification + skilled personnel Conversion of the viruses really counted to viruses per ml BS: burst size, the numbers of viruses liberated by lytic events FVIC: the % of bacterial cells visibly infected by viruses FIC: the % of bacterial cells infected VIBM: the % of bacterial production removal due to viral lysis

12 FCM: Flow cytometry Viruses Bacteria Type: Cytometry Principle: Target Bacterial and viral DNA fluorescence after laser excitation. DNA fluorescence is obtained with a highly fluorescent acid nucleic dye (SYBR Green I for example). Counts: device counts and software analysis

13 Advantages Disadvantages Quick method Useful for routine quantification Reproducible Do not directly see the organisms Over-estimation of viruses? Particle size and fluorescence: limit of cytometry detection

14 Comparison EFM / TEM / FCM Bettarel Y., T. Sime-Ngando, C. Amblard and H. Laveran (2000). A comparison of methods for counting viruses in aquatic systems. Applied and Environmental Microbiology 66(6): Marie D., C. P. D. Brussaard, R. Thyrhaug, G. Bratbak and D. Vaulot (1999). Enumeration of marine viruses in culture and natural samples by flow cytometry. Applied and Environmental Microbiology 65(1): FCM counts 6.E+06 4.E+06 1:1 Weimbauer M.G., and T. Suttle. (1997). Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Aquatic Microbial Ecology 13: E+06 0.E E E E E+06 EFM Counts Virus-like particles counts can be significantly different Difficulty to compare results obtained with different methods

15 10 Annecy Bourget Geneva A B C Accessing viral dynamics Flow cytometry Depth (m) P icocyano (cell.m l -1 ) 1e+2 1e+3 1e+4 1e+5 1e+6 picoyanobacteria 10 D E F Depth (m) S m all eukaryotes (cell.m l -1 ) 1e+2 1e+3 1e+4 1e+5 1e+6 small eukaryotes 10 G H I Depth (m) Depth (m) H et. bacteria (cell.m l -1 ) J Data not available K L 1e+6 2e+6 3e+6 4e+6 5e+6 heterotrophic bacteria viruses 40 5e+7 1e+8 Virus (part.ml -1 ) 50 Aug Dec Apr Aug Dec Aug Dec Apr Aug Dec 1.5e+8 2e+8 Aug Dec Apr Aug Dec

16 Accessing viral-induced induced bacterial mortality Microcosms. DialysisBags. Bottles Agents of bacterial mortality % of bacterial mortality due to the viral lytic activity Exp. Enrichment Exp. Dilution Direct? Not Direct? TEM Dilution technique (FCM counts)

17 Accessing virus-induced induced bacterial mortality Lake water ultrafiltrate (0.02 µm) 0% Control 20% 40% 70% 100% Grazer free Lake water Illustration of the dilution results y = -0,0045x + 0,8948 Bacterial Growth rate (d -1 ) R 2 = 0,9397 Dilution technique Dilution Contact rates bacteria/viruses

18 Accessing viral-induced induced bacterial mortality Heterotrophic Bacterial Mortality Viral lyses (TEM) Viral lyses (Dilution/FCM) Flagellates Grazing EXP. Lake Geneva May %.d -1 9 %.d %.d -1 EXP. Lake Le Bourget April %.d %.d %.d -1 EXP. Lake Le Bourget May %.d %.d %.d -1 EXP. Lake Le Bourget August %.d %.d %.d -1

19 Techniques for viral counts may give different results Techniques to asses viral-induced induced bacterial mortality may also give different results We have to keep this in mind before making reliable comparisons between ecosystems ---- Hence, the question is asked about the possibility of a common procedure or any universal way of counting and assessing mortality processes A future objective: European data basis / inter-calibration of methods? No evident answer yet and will be there any? Conclusion

1 st European Workshop on Aquatic Phage Ecology Castle of Ripaille, Thonon-les-Bains, 2-4 February 2005 Abstract of the proposed workshop topic: Viruses infecting bacteria and/or cyanobacteria are an

20 1 st European Workshop on Aquatic Phage Ecology Castle of Ripaille, Thonon-les-Bains, 2-4 February 2005 Abstract of the proposed workshop topic: Viruses infecting bacteria and/or cyanobacteria are an essential biological compartment in aquatic microbial food webs. They are important controlling agents for planktonic communities, playing a key role in cell mortality, nutrient cycles, microbial diversification and diversity. However, still little is known on how this viral activity is linked to diversity and ecosystem functioning. Organization: Stéphan JACQUET INRA This first workshop in Europe will provide an opportunity for promoting this field of research through discussion and presentation of our past and most recent results, of the various approaches and methods we use and the problems we face, for reinforcing or developing collaboration, and in fine for developing strategies for future research.

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